Anti-ngf antibodies and their use

ABSTRACT

The present disclosure encompasses NGF binding proteins, specifically to antibodies that are chimeric, CDR grafted and canonized antibodies, and methods of making and uses thereof. The antibodies, or antibody portions, of the disclosure are useful for detecting NGF and for inhibiting NGF activity, e.g., in a mammal subject suffering from a disorder in which NGF activity is detrimental.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of currently pending U.S. applicationSer. No. 13/817,721, which is a national stage 371 application of theinternational application PCT/US2011/048518 filed on Aug. 19, 2011 whichclaims priority to the U.S. Provisional Application No. 61/375,193,filed Aug. 19, 2010, all contents of which are incorporated by referencein their entirety

TECHNICAL FIELD

The disclosure relates to anti-NGF antibodies and polynucleotidesencoding the same, and use of such antibodies and/or polynucleotides inthe treatment and/or prevention of pain, including but not limited topost-surgical pain, rheumatoid arthritis pain, cancer pain, andosteoarthritis pain.

BACKGROUND

Nerve growth factor (NGF) is a secreted protein that was discovered over50 years ago as a molecule that promotes the survival anddifferentiation of sensory and sympathetic neurons. (SeeLevi-Montalcini, Science 187: 113 (1975), for a review). The crystalstructure of NGF and NGF in complex with the tyrosine kinase A (TrkA)receptor has been determined (McDonald et al., Nature 354: 411 (1991);Wiesmann et al., Nature 401: 184-188 (1999)).

The role of NGF in the development and survival of both peripheral andcentral neurons has been well characterized. NGF has been shown to be acritical survival and maintenance factor in the development ofperipheral sympathetic and embryonic sensory neurons and of basalforebrain cholinergic neurons (see, e.g., Smeyne et al., Nature 368:246-9 (1994); and Crowley et al., Cell, 76: 1001-11 (1994)). It has beenshown to inhibit amyloidogenesis that leads to Alzheimer's disease(Calissano et al., Cell Death and Differentiation, 17: 1126-1133(2010)). NGF up-regulates expression of neuropeptides in sensory neurons(Lindsay et al., Nature, 337:362-364 (1989)) and its activity ismediated through two different membrane-bound receptors, the TrkAreceptor and the p75 common neurotrophin receptor (Chao et al., Science,232:518-521 (1986); Huang et al., Annu. Rev. Neurosci., 24:677-736(2001); Bibel et al., Genes Dev., 14:2919-2937 (2000)).

NGF is produced by a number of cell types including mast cells (Leon, etal., Proc. Natl. Acad. Set, 91: 3739-3743 (1994)), B-lymphocytes(Torcia, et al., Cell, 85: 345-356 (1996), keratinocytes (Di Marco, etal., J. Biol. Chem., 268: 22838-22846)), smooth muscle cells (Ueyama, etal., J. Hypertens., 11: 1061-1065 (1993)), fibroblasts (Lindholm, etal., Eur. J. Neurosci., 2: 795-801 (1990)), bronchial epithelial cells(Kassel, et al., Clin, Exp. Allergy, 31: 1432-40 (2001)), renalmesangial cells (Steiner, et al., Am. J. Physiol., 261:F792-798 (1991))and skeletal muscle myotubes (Schwartz, et al., J Photochem. Photobiol.,B66: 195-200 (2002)). In addition, NGF receptors have been found on avariety of cell types outside of the nervous system.

NGF has been implicated in processes outside of the nervous system,e.g., NGF has been shown to enhance vascular permeability (Otten, etal., Eur J Pharmacol., 106: 199-201 (1984)), enhance T- and B-cellimmune responses (Otten, et al., Proc. Natl. Acad. Sci., USA 86:10059-10063 (1989)), induce lymphocyte differentiation and mast cellproliferation and cause the release of soluble biological signals frommast cells (Matsuda, et al., Proc. Natl. Acad. Sci., 85: 6508-6512(1988); Pearce, et al., J. Physiol, 372:379-393 (1986); Bischoff, etal., Blood, 79: 2662-2669 (1992); Horigome, et al., J. Biol. Chem., 268:14881-14887 (1993)).

Both local and systemic administrations of NGF have been shown to elicithyperalgesia and allodynia (Lewin, G. R. et al., Eur. J. Neurosci. 6:1903-1912 (1994)). Intravenous infusion of NGF in humans produces awhole body myalgia while local administration evokes injection sitehyperalgesia and allodynia in addition to the systemic effects (Apfel,S. C. et al., Neurology, 51: 695-702(1998)). Furthermore, in certainforms of cancer, excess NGF facilitates the growth and infiltration ofnerve fibers with induction of cancer pain (Zhu, Z. et al., J Clin.Oncol., 17: 241-228 (1999). Although exogenously added NGF has beenshown to be capable of having all of these effects, it is important tonote that it has only rarely been shown that endogenous NGF is importantin any of these processes in vivo (Torcia, et al., Cell, 85(3): 345-56(1996)).

An elevated level of NGF has been implicated in certain inflammatoryconditions in humans and animals, e.g., systemic lupus erythematosus(Bracci-Laudiero, et al., Neuroreport, 4: 563-565 (1993)), multiplesclerosis (Bracci-Laudiero, et al., Neurosci. Lett., 147:9-12 (1992)),psoriasis (Raychaudhuri, et al., Acta Derm. Venereol, 78: 84-86 (1998)),arthritis (Falcim, et al., Ann. Rheum. Dis., 55: 745-748 (1996)),interstitial cystitis (Okragly, et al., J. Urology 6: 438-441 (1999))and asthma (Braun, et al., Eur. J Immunol., 28:3240-3251 (1998)). Thesynovium of patients affected by rheumatoid arthritis expresses highlevels of NGF while in non-inflamed synovium NGF has been reported to beundetectable (Aloe, et al., Arch. Rheum., 35:351-355 (1992)). Similarresults were seen in rats with experimentally induced rheumatoidarthritis (Aloe, et al., Clin. Exp. Rheumatol., 10: 203-204 (1992)).Elevated levels of NGF have been reported in transgenic arthritic micealong with an increase in the number of mast cells (Aloe, et al., Int.J. Tissue Reactions-Exp. Clin. Aspects, 15: 139-143 (1993)).Additionally, elevated levels of expression of canine NGF has been shownin lame dogs (Isola, M., Ferrari, V., Stabile, F., Bemardini, D.,Gamier, P., Busetto, R. Nerve growth factor concentrations in thesynovial fluid from healthy dogs and dogs with secondary osteoarthritis.Vet. Comp. Orthop. Traumatol. 4: 279 (2011)). PCT Publication No. WO02/096458 discloses use of anti-NGF antibodies of certain properties intreating various NGF related disorders such as inflammatory condition(e.g., rheumatoid arthritis). It has been reported that a purifiedanti-NGF antibody injected into arthritic transgenic mice carrying thehuman tumor necrosis factor (TNF) gene caused reduction in the number ofmast cells, as well as a decrease in histamine and substance P levelswithin the synovium of arthritis mice (Aloe et al., Rheumatol. Int., 14:249-252 (1995)). It has been shown that exogenous administration of aNGF antibody reduced the enhanced level of TNF occurring in arthriticmice (Manni et al., Rheumatol. Int., 18: 97-102 (1998)).

Increased expression of NGF and high affinity NGF receptor (TrkA) wasobserved in human osteoarthritis chondrocytes (Iannone et al.,Rheumatology, 41: 1413-1418 (2002)). Rodent anti-NGF antagonistantibodies have been reported (Hongo et al., Hybridoma, 19(3):215-227(2000); Ruberti et al., Cell. Molec. Neurobiol., 13(5): 559-568 (1993)).However, when rodent antibodies are used therapeutically in non-rodentsubjects, an anti-murine antibody response develops in significantnumbers of treated subjects.

The involvement of NGF in chronic pain has led to considerable interestin therapeutic approaches based on inhibiting the effects of NGF(Saragovi, et al., Trends Pharmacol Sci. 21: 93-98 (2000)). For example,a soluble form of the TrkA receptor was used to block the activity ofNGF, which was shown to significantly reduce the formation of neuromas,responsible for neuropathic pain, without damaging the cell bodies ofthe lesioned neurons (Kryger, et al., J. Hand Surg. (Am.), 26: 635-644(2001)).

Certain anti-NGF antibodies have been described (PCT Publication Nos. WO2001/78698, WO 2001/64247, WO 2002/096458, WO 2004/032870, WO2005/061540, WO 2006/131951, WO 2006/110883; U.S. Publication Nos. US20050074821, US 20080033157, US 20080182978 and US 20090041717; and U.S.Pat. No. 7,449,616). In animal models of neuropathic pain (e.g., nervetrunk or spinal nerve ligation) systemic injection of neutralizingantibodies to NGF prevents both allodynia and hyperalgesia (Ramer etal., Eur. J. Neurosci., 11: 837-846 (1999); Ro et al., Pain, 79: 265-274(1999)). Furthermore, treatment with a neutralizing anti-NGF antibodyproduces significant pain reduction in a murine cancer pain model(Sevcik et al., Pain, 115: 128-141 (2005)). Thus, there is a seriousneed for anti-NGF antagonist antibodies for humans and animals.

SUMMARY OF THE INVENTION

The present disclosure provides a novel family of binding proteins, CDRgrafted antibodies, mammalized (such as bovanized, camelized, caninized,equinized, felinized, humanized etc.) antibodies, and fragments thereof,capable of binding and neutralizing NGF. The disclosure provides atherapeutic means with which to inhibit NGF and provides compositionsand methods for treating disease associated with increased levels ofNGF, particularly inflammatory disorders.

In one aspect, the present disclosure provides a binding protein, orfragment thereof, comprising hypervariable region sequences wholly orsubstantially identical to sequences from an antibody from a donorspecies; and constant region sequences wholly or substantially identicalto sequences of antibodies from a target species, wherein the donor andtarget species are different. The binding protein may for examplespecifically bind NGF and have a heavy chain having a heavy chainvariable region and a light chain having a light chain variable region.

In another aspect, the present disclosure provides a binding proteinthat specifically binds NGF and which has a heavy chain having a heavychain variable region and a light chain having a light chain variableregion, wherein the heavy chain variable region comprises an amino acidsequence having at least 90% identity with a sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ IDNO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO:166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In another aspect, the present disclosure provides a binding proteinthat specifically binds NGF and which has a heavy chain having a heavychain variable region and a light chain having a light chain variableregion, wherein the light chain variable region comprises an amino acidsequence having at least 90% identity with a sequence selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ IDNO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ IDNO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176,SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ IDNO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198,SEQ ID NO: 200, SEQ ID NO: 202, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

A binding protein of the present disclosure may comprise at least oneCDR comprising an amino acid sequence selected from: a) heavy chain CDRsconsisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75,79, 80, 81, and modified CDR amino acid sequences having a sequenceidentity of at least 50% to one of said sequences; and b) light chainCDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76,77, 78, 82, 83, 84, and modified CDR amino acid sequences having asequence identity of at least 50% to one of said sequences.Alternatively, the binding protein of the present disclosure maycomprise at least one CDR comprising an amino acid sequence selectedfrom: a) heavy chain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62,63, 67, 68, 69, 73, 74, 75, 79, 80, 81, and modified CDR amino acidsequences having a sequence identity of at least 70% to one of saidsequences; and b) light chain CDRs consisting of SEQ ID NO: 58, 59, 60,64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84, and modified CDR aminoacid sequences having a sequence identity of at least 70% to one of saidsequences. The binding protein of the present disclosure may comprise atleast one CDR comprising an amino acid sequence selected from: a) heavychain CDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69,73, 74, 75, 79, 80, 81, and modified CDR amino acid sequences having asequence identity of at least 80% to one of said sequences; and b) lightchain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72,76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having asequence identity of at least 80% to one of said sequences, bindingprotein of the present disclosure may comprise at least one CDRcomprising an amino acid sequence selected from: a) heavy chain CDRsconsisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75,79, 80, 81, and modified CDR amino acid sequences having a sequenceidentity of at least 90% to one of said sequences; and b) light chainCDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76,77, 78, 82, 83, 84, and modified CDR amino acid sequences having asequence identity of at least 90% to one of said sequences.

A binding protein of the present disclosure may comprise a heavy chainhuman immunoglobulin constant domain selected from the group consistingof IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgEconstant domain, IgG2 constant domain, IgG3 constant domain, and IgAconstant domain. A binding proteins of the present disclosure mayalternatively comprise a heavy chain canine immunoglobulin constantdomain selected from the group consisting of IgM constant domain, IgG4constant domain, IgG1 constant domain, IgE constant domain, IgG2constant domain, IgG3 constant domain, and IgA constant domain. Abinding protein of the present disclosure may alternatively comprise aheavy chain feline immunoglobulin constant domain. A binding protein ofthe present disclosure may alternatively comprise a heavy chain equineimmunoglobulin constant domain. A binding protein of the presentdisclosure may further comprise a constant region having an amino acidsequence selected from the group consisting of SEQ ID NO: 52 and SEQ IDNO: 54.

Any of the above binding proteins may be selected from the groupconsisting of; an immunoglobulin molecule, disulfide linked Fv,monoclonal antibody, scFv, chimeric antibody, single domain antibody,CDR-grafted antibody, diabody, humanized antibody, caninized mAb, caninemAb, feline mAb, felinized mAb, equine mAb, equinized mAb, amultispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, aFab′, a bispecific antibody, a F(ab′)2, and a Fv.

Any of the above binding proteins may be capable of modulating abiological function of NGF, or neutralizing NGF.

Any of the above binding proteins may be capable of neutralizing NGFwith a potency (IC₅₀) of at least about 10 nM, at least about 5 nM, atleast about 1 nM, at least about 0.5 nM, at least about 0.1 nM, at leastabout 0.05 nM, at least about 0.01 nM, or at least about 0.001 nM, asmeasured in the TF-1 cell proliferation assay or the pERK and Pathhunterassays.

Any of the above binding proteins may have an on rate constant (K_(on))for NGF of: at least about 10² M⁻¹s⁻¹, at least about 10³ M⁻¹s⁻¹, atleast about M⁻¹s⁻¹, at least about 10⁵ M⁻¹s⁻¹, or at least about 10⁶M⁻¹s⁻¹, or at least about 10⁷ M⁻¹s⁻¹, as measured by surface plasmonresonance.

Any of the above binding proteins may have an off rate constant(K_(off)) for NGF selected from the group consisting of: at most about10⁻³ s⁻¹, at most about 10⁻⁴ s⁻¹, at most about 10⁻⁵ s⁻¹, at most about10⁻⁶ s⁻¹, and at most about 10⁻⁷ s⁻¹, as measured by surface plasmonresonance.

Any of the above binding proteins may have a dissociation constant(K_(D)) for NGF selected from the group consisting of: at most about10⁻⁷ M, at most about 10⁻⁸ M, at most about 10⁻⁹ M, at most about 10⁻¹⁰M, at most about 10⁻¹¹ M at most about 10⁻¹² M, at most about 10⁻¹³ Mand at most about 10⁻¹⁴ M. The dissociation constant (K_(D)) may be, forexample, about 1×10⁻⁹ M, about 1×10⁻¹⁰ M, about 3.14×10⁻¹⁰M, about1×10⁻¹¹ M, about 2.37×10⁻¹¹ M, about 1×10⁻¹² M, about 1×10⁻¹³ M, andabout 3.3×10⁻¹⁴ M.

Any of the above binding proteins may further comprise an agent selectedfrom the group consisting of; an immunoadhension molecule, an imagingagent, a therapeutic agent, and a cytotoxic agent. The agent may be, forexample, an imaging agent selected from the group consisting of aradiolabel, an enzyme, a fluorescent label, a luminescent label, abioluminescent label, a magnetic label, and biotin. The imaging agentmay be a radiolabel selected from the group consisting of: 3H, 14C, 35S,90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm. Alternatively,the agent may be a therapeutic or cytotoxic agent, such as, for example,an anti-metabolite, an alkylating agent, an antibiotic, a growth factor,a cytokine, an anti-angiogenic agent, an anti-mitotic agent, ananthracycline, toxin, and an apoptotic agent.

Any of the binding proteins may possess a murine, canine, feline, humanor equine glycosylation pattern.

Any of the binding proteins may be a crystallized binding protein. Thecrystallized binding protein may be a carrier-free pharmaceuticalcontrolled release crystallized binding protein.

In another aspect, the present disclosure provides an isolated nucleicacid encoding any of the above binding proteins. The isolated nucleicacid may comprise RNA or DNA.

In another aspect, the present disclosure provides an isolated nucleicacid comprising or complementary to a nucleic acid sequence that encodesa binding protein that specifically binds NGF having a heavy chainhaving a heavy chain variable region and a light chain having a lightchain variable region, wherein the heavy chain variable region isencoded by a nucleotide sequence having at least 90% sequence identitywith a sequence selected from the group consisting of SEQ ID NOs: 1, 5,9, 13, 17, and 21.

In another aspect, the present disclosure provides an isolated nucleicacid comprising or complementary to a nucleic acid sequence that encodesa binding protein that specifically binds NGF having a heavy chainhaving a heavy chain variable region and a light chain having a lightchain variable region, wherein the light chain variable region isencoded by a nucleotide sequence having at least 90% sequence identitywith a sequence selected from the group consisting of SEQ ID NOS: 3, 7,11, 15, 19 and 23.

In another aspect, the present disclosure provides a recombinant vectorcomprising an isolated nucleic acid encoding a binding protein thatspecifically binds NGF as described herein. A recombinant vectoraccording to the present disclosure may comprise pcDNA, pTT, pTT3,pEFBOS, pBV, pJV or pBJ. Also provided is a host cell comprising such arecombinant vector. The host cell may be for example a eukaryotic cell,or a prokaryotic cell. The host cell may be a protist cell; an animalcell such as but not limited to a mammalian cell, avian cell; an insectcell, such as but not limited to an insect Sf9 cell; a plant cell; or afungal cell. The host cell may be for example an E. coli cell. The hostcell may be a CHO cell, or a COS cell. Also provided is an isolated cellline that produces a binding protein that specifically binds NGF asdescribed herein.

In another aspect, the present disclosure provides a pharmaceutical ordiagnostic composition comprising a binding protein that specificallybinds NGF as described herein, and a pharmaceutically acceptablecarrier, diluent or excipient. A pharmaceutical composition may comprisea therapeutically effective amount of the NGF binding protein.

In another aspect, the present disclosure provides a composition for therelease of a binding protein, the composition comprising: (a) acomposition comprising a binding protein that specifically binds NGF asdescribed herein, and a pharmaceutically acceptable carrier, excipientor diluent, and (b) at least one polymeric carrier.

In another aspect, the present disclosure provides a method for reducingNGF activity in a subject (for example, a dog, cat, horse, ferret, etc.)suffering from a disorder in which NGF activity is detrimental,comprising administering to the subject a therapeutically effectiveamount of a binding protein that specifically binds NGF as describedherein.

In another aspect, the present disclosure provides a method for makinganti-NGF antibodies comprising: (a) production of murine monoclonalantibodies; (b) screening hybridoma supernatants; (c) grafting of donorCDRs into target frameworks; and (d) introducing backmutations in theframework region of the target antibodies, wherein the anti-NGFantibodies comprise hypervariable region sequences wholly orsubstantially identical to sequences from an antibody from the donorspecies and constant region sequences wholly or substantially identicalto sequences of an antibody from the target species, wherein the donorand the target species are different. In the method, the donor may be,for example, a mouse and the target a non-murine mammal, such as but notlimited to a bovine, canine, equine, or feline mammal, or a camel goat,human or sheep.

In another aspect, the present disclosure provides a method fordetecting the presence or amount of NGF in a sample, comprising:providing a reagent comprising any of the above binding proteins thatspecifically bind NGF; combining the binding protein with the sample fora time and under conditions sufficient for the binding protein to bindto any NGF in the sample; and determining the presence or amount of NGFin the sample based on specific binding of the binding protein to NGF.In the method, the binding protein may be immobilized or may be capableof being immobilized on a solid support. In the method, the bindingprotein may be coupled to a detectable label, such as, for example, animaging agent such as but not limited to a radiolabel, an enzyme, afluorescent label, a luminescent label, a bioluminescent label, amagnetic label, and biotin. The imaging agent may be for example aradiolabel selected from the group consisting of: 3H, 14C, 35S, 90Y,99Tc, 111 In, 125I, 131I, ¹⁷⁷Lu, 166Ho, and 153Sm.

In another aspect, the present disclosure provides an immunoassay devicefor detecting the presence or amount of NGF in a sample, the devicecomprising any of the above binding proteins that specifically bind NGF,immobilized on a solid support.

In another aspect, the present disclosure provides a kit for detectingthe presence or amount of NGF in a sample, the kit comprising: animmunoreagent comprising any of the above binding proteins thatspecifically bind NGF and instructions for determining the presence oramount of NGF in the sample based on specific binding of theimmunoreagent to NGF. In the kit, the binding protein may be immobilizedon a solid support.

In still yet another aspect, the present disclosure relates to anantibody or antigen binding fragment thereof comprising:

a heavy chain variable region comprises an amino acid sequence having atleast 90% identity with a sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18,SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167,SEQ ID NO:168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ IDNO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185,SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ IDNO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203,SEQ ID NO: 206, SEQ ID NO: 207, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof; and

a light chain variable region comprises an amino acid sequence having atleast 90% identity with a sequence selected from the group consisting ofSEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20,SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ IDNO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO:173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO:193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQID NO: 202, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

More specifically, the above-described antibody may comprise at leastone CDR comprising an amino acid sequence selected from: a) heavy chainCDRs consisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73,74, 75, 79, 80, 81, and modified CDR amino acid sequences having asequence identity of at least 50% to one of said sequences; and b) lightchain CDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72,76, 77, 78, 82, 83, 84, and modified CDR amino acid sequences having asequence identity of at least 50% to one of said sequences.Alternatively, the above-described antibody may comprise at least oneCDR comprising an amino acid sequence selected from: a) heavy chain CDRsconsisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75,79, 80, 81, and modified CDR amino acid sequences having a sequenceidentity of at least 70% to one of said sequences; and b) light chainCDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76,77, 78, 82, 83, 84, and modified CDR amino acid sequences having asequence identity of at least 70% to one of said sequences.Alternatively, the above-described antibody may comprise at least oneCDR comprising an amino acid sequence selected from: a) heavy chain CDRsconsisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75,79, 80, 81, and modified CDR amino acid sequences having a sequenceidentity of at least 80% to one of said sequences; and b) light chainCDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76,77, 78, 82, 83, 84, and modified CDR amino acid sequences having asequence identity of at least 80% to one of said sequences.Alternatively, the above-described antibody may comprise at least oneCDR comprising an amino acid sequence selected from: a) heavy chain CDRsconsisting of SEQ ID NO: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74, 75,79, 80, 81, and modified CDR amino acid sequences having a sequenceidentity of at least 90% to one of said sequences; and b) light chainCDRs consisting of SEQ ID NO: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76,77, 78, 82, 83, 84, and modified CDR amino acid sequences having asequence identity of at least 90% to one of said sequences.

The above-described antibody may comprise a heavy chain humanimmunoglobulin constant domain selected from the group consisting of IgMconstant domain, IgG4 constant domain, IgG1 constant domain, IgEconstant domain, IgG2 constant domain, IgG3 constant domain, and IgAconstant domain. More specifically, the antibody may comprise a heavychain canine immunoglobulin constant domain selected from the groupconsisting of IgM constant domain, IgG4 constant domain, IgG1 constantdomain, IgE constant domain, IgG2 constant domain, IgG3 constant domain,and IgA constant domain. Alternatively, the antibody comprises a heavychain feline immunoglobulin constant domain. Still furtheralternatively, the antibody comprises a heavy chain equineimmunoglobulin constant domain. Moreover, the above-described antibodymay comprise a constant region having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Stillfurther, the above-described antibody is selected from the groupconsisting of: an immunoglobulin molecule, disulfide linked Fv,monoclonal antibody, scFv, chimeric antibody, single domain antibody,CDR-grafted antibody, diabody, humanized antibody, caninized mAb, caninemAb, feline mAb, felinized mAb, equine mAb, equinized mAb, amultispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, aFab′, a bispecific antibody, a F(ab′)₂, and a Fv.

In another aspect, the above-identified antibody is capable ofmodulating a biological function of NGF.

In still yet another aspect, the present disclosure relates to anisolated nucleic acid encoding the above-described antibody.

In another aspect, the present invention relates to an antibody orantigen binding fragment thereof having a heavy chain variable regionthat comprises an amino acid sequence having at least 90% identity witha sequence of SEQ ID NO:37 and a light chain variable region thatcomprises an amino acid sequence having at least 90% identity with asequence of SEQ ID NO:38. The above-described antibody may comprise aheavy chain human immunoglobulin constant domain selected from the groupconsisting of IgM constant domain, IgG4 constant domain, IgG1 constantdomain, IgE constant domain, IgG2 constant domain, IgG3 constant domain,and IgA constant domain. More specifically, the antibody may comprise aheavy chain canine immunoglobulin constant domain selected from thegroup consisting of IgM constant domain, IgG4 constant domain, IgG1constant domain, IgE constant domain, IgG2 constant domain, IgG3constant domain, and IgA constant domain. Alternatively, the antibodycomprises a heavy chain feline immunoglobulin constant domain. Stillfurther alternatively, the antibody comprises a heavy chain equineimmunoglobulin constant domain. Moreover, the above-described antibodymay comprise a constant region having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Stillfurther, the above-described antibody is selected from the groupconsisting of: an immunoglobulin molecule, disulfide linked Fv,monoclonal antibody, scFv, chimeric antibody, single domain antibody,CDR-grafted antibody, diabody, humanized antibody, caninized mAb, caninemAb, feline mAb, felinized mAb, equine mAb, equinized mAb, amultispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, aFab′, a bispecific antibody, a F(ab′)₂, and a Fv.

In another aspect, the above-identified antibody is capable ofmodulating a biological function of NGF.

In still yet another aspect, the present disclosure relates to anisolated nucleic acid encoding the above-described antibody.

In another aspect, the present invention relates to an antibody orantigen binding fragment thereof having a heavy chain variable regioncomprises an amino acid sequence having at least 90% identity with asequence of SEQ ID NO: 192 and the light chain variable region comprisesan amino acid sequence having at least 90% identity with a sequence ofSEQ ID NO: 193. The above-described antibody may comprise a heavy chainhuman immunoglobulin constant domain selected from the group consistingof IgM constant domain, IgG4 constant domain, IgG1 constant domain, IgEconstant domain, IgG2 constant domain, IgG3 constant domain, and IgAconstant domain. More specifically, the antibody may comprise a heavychain canine immunoglobulin constant domain selected from the groupconsisting of IgM constant domain, IgG4 constant domain, IgG1 constantdomain, IgE constant domain, IgG2 constant domain, IgG3 constant domain,and IgA constant domain. Alternatively, the antibody comprises a heavychain feline immunoglobulin constant domain. Still furtheralternatively, the antibody comprises a heavy chain equineimmunoglobulin constant domain. Moreover, the above-described antibodymay comprise a constant region having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 52 and SEQ ID NO: 54. Stillfurther, the above-described antibody is selected from the groupconsisting of: an immunoglobulin molecule, disulfide linked Fv,monoclonal antibody, scFv, chimeric antibody, single domain antibody,CDR-grafted antibody, diabody, humanized antibody, caninized mAb, caninemAb, feline mAb, felinized mAb, equine mAb, equinized mAb, amultispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, aFab′, a bispecific antibody, a F(ab′)₂, and a Fv.

In another aspect, the above-identified antibody is capable ofmodulating a biological function of NGF.

In still yet another aspect, the present disclosure relates to anisolated nucleic acid encoding the above-described antibody.

In still yet another aspect, the present disclosure relates to apharmaceutical or diagnostic composition comprising at least one of theabove-described antibodies, and a pharmaceutically acceptable carrier,diluent or excipient. More specifically, the pharmaceutical ordiagnostic composition may comprise a therapeutically effective amountof at least one of the above-described antibodies. In addition, thepharmaceutical or diagnostic composition may comprise at onepreservative. An example of at least one preservative that may be usedis methylparaben, propylparaben, benzyl alcohol, chlorobutanol orbenzalkonium chloride.

The pharmaceutical composition can have a pH of greater than about 7.0.Alternatively, the pharmaceutical composition can have a pH of betweenabout 6.8 and about 8.2. Alternatively; the pharmaceutical compositioncan have a pH of between about 7.2 and about 7.8. Still furtheralternatively, the pH of the pharmaceutical composition can be betweenabout 7.4 and about 7.6. Still further alternatively, the pH of thepharmaceutical composition can be about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 or 8.2.

The pharmaceutical composition of the present disclosure may have ahalf-life of from about 8.0 days to about 15.0 days when dosedintravenously or subcutaneously. Alternatively, the pharmaceuticalcomposition of the present invention may have a half-life of from about10.0 days to about 13.0 days. Still further alternatively, thepharmaceutical composition of the present invention may have a half-lifeof about 8.0 days, about 8.5 days, about 9.0 days, about 9.5 days, about10.0 days, about 10.5 days, about 11.0 days, about 11.5 days, about 12.0days, about 12.5 days, about 13.0 days, about 13.5 days, about 14.0days, about 14.5 days or about 15.0 days.

In another aspect, the present disclosure relates to a method forreducing NGF activity in a subject suffering from a disorder in whichNGF activity is detrimental, comprising administering to the subject atherapeutically effective amount of an antibody of antigen bindingfragment thereof of at least one of the above-described antibodies orantigen-binding fragments thereof.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates PR-1254972 VH nucleotide sequence (SEQ ID NO: 1) ofmouse anti-NGF antibody.

FIG. 2 illustrates PR-1254972 VH amino acid sequence (SEQ ID NO: 2) ofmouse anti-NGF antibody.

FIG. 3 illustrates PR-1254972 VL nucleotide sequence (SEQ ID NO: 3) ofmouse anti-NGF antibody.

FIG. 4 illustrates PR-1254972 VL amino acid (SEQ ID NO: 4) of mouseanti-NGF antibody.

FIG. 5 illustrates PR-1254973 VH nucleotide sequence (SEQ ID NO: 5) ofmouse anti-NGF antibody.

FIG. 6 illustrates PR-1254973 VH amino acid (SEQ ID NO: 6) of mouseanti-NGF antibody.

FIG. 7 illustrates PR-1254973 VL nucleotide sequence (SEQ ID NO: 7) ofmouse anti-NGF antibody.

FIG. 8 illustrates PR-1254973 VL amino acid (SEQ ID NO: 8) of mouseanti-NGF antibody.

FIG. 9 illustrates PR-1254977 VH nucleotide sequence (SEQ ID NO: 9) ofmouse anti-NGF antibody.

FIG. 10 illustrates PR-1254977 VH amino acid (SEQ ID NO: 10) of mouseanti-NGF antibody.

FIG. 11 illustrates PR-1254977 VL nucleotide sequence (SEQ ID NO: 11) ofmouse anti-NGF antibody.

FIG. 12 illustrates PR-1254977 VL amino acid (SEQ ID NO: 12) of mouseanti-NGF antibody.

FIG. 13 illustrates PR-1254980 VH nucleotide sequence (SEQ ID NO: 13) ofmouse anti-NGF antibody.

FIG. 14 illustrates PR-1254980 VH amino acid (SEQ ID NO: 14) of mouseanti-NGF antibody.

FIG. 15 illustrates PR-1254980 VL nucleotide sequence (SEQ ID NO: 15) ofmouse anti-NGF antibody.

FIG. 16 illustrates PR-1254980 VL amino acid (SEQ ID NO: 16) of mouseanti-NGF antibody.

FIG. 17 illustrates PR-1254981 VH nucleotide sequence (SEQ ID NO: 17) ofmouse anti-NGF antibody.

FIG. 18 illustrates PR-1254981 VH amino acid (SEQ ID NO: 18) of mouseanti-NGF antibody.

FIG. 19 illustrates PR-1254981 VL nucleotide sequence (SEQ ID NO: 19) ofmouse anti-NGF antibody.

FIG. 20 illustrates PR-1254981 VL amino acid (SEQ ID NO: 20) of mouseanti-NGF antibody.

FIG. 21 illustrates PR-1254982 VH nucleotide sequence (SEQ ID NO: 21) ofmouse anti-NGF antibody.

FIG. 22 illustrates PR-1254982 VH amino acid (SEQ ID NO: 22) of mouseanti-NGF antibody.

FIG. 23 illustrates PR-1254982 VL nucleotide sequence (SEQ ID NO: 23) ofmouse anti-NGF antibody.

FIG. 24 illustrates PR-1254982 VL amino acid (SEQ ID NO: 24) of mouseanti-NGF antibody.

FIG. 25 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 25 (72.1 VH aminoacid).

FIG. 26 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 26 (72.1 VL aminoacid).

FIG. 27 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined) SEQ ID NO: 27 (73.1 VH aminoacid).

FIG. 28 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined) SEQ ID NO: 28 (73.1 VL aminoacid).

FIG. 29 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 29 (77.1 VH aminoacid).

FIG. 30 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 30 (77.1 VL aminoacid).

FIG. 31 A illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 31 (81.1 VH aminoacid).

FIG. 31 B illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 177 (81.1B VHamino acid).

FIG. 32 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 32 (81.1 VL aminoacid)

FIG. 33 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 33 (82.1 VH aminoacid)

FIG. 34 illustrates mouse anti-NGF mAb caninized by CDR grafting ontocanine Ig frameworks (CDRs are underlined), SEQ ID NO: 34 (82.1 VL aminoacid).

FIG. 35 illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 35(72.2 VH amino acid).

FIG. 36A illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 36(72.2 VL amino acid).

FIG. 36B illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 179(72.3 VH amino acid).

FIG. 36C illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 180(72.4 VH amino acid).

FIG. 36D illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 181(72.4 VL amino acid).

FIG. 37 illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 37(73.2 VH amino acid).

FIG. 38A illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 38(73.2 VL amino acid).

FIG. 38B illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 182(73.4 VH amino acid).

FIG. 38C illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 183(73.4 VL amino acid).

FIG. 39 illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 39(77.2 VH amino acid).

FIG. 40A illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 40(77.2 VL amino acid).

FIG. 40B illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 184(77.3 VH amino acid).

FIG. 40C illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 185(77.4 VH amino acid).

FIG. 40D illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 186(77.4 VL amino acid).

FIG. 41 illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 41(81.2 VH amino acid).

FIG. 42A illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 42(81.2 VL amino acid).

FIG. 42B illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 187(81.4 VH amino acid).

FIG. 42C illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 188(81.4 VL amino acid).

FIG. 42D illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 189(81.2B VH amino acid).

FIG. 42E illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 190(81.4B VH amino acid).

FIG. 42F illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold, SEQ ID NO: 206(81.5B VH amino acid).

FIG. 42G illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold, SEQ ID NO: 207(81.6B VH amino acid).

FIG. 43 illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 43(82.2 VH amino acid).

FIG. 44A illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 44(82.2 VL amino acid).

FIG. 44B illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 191(82.3 VL amino acid).

FIG. 44C illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 192(82.4 VH amino acid).

FIG. 44D illustrates caninized anti-NGF antibodies containing backmutation residues (backmutation residues shown in bold), SEQ ID NO: 193(82.4 VL amino acid).

FIG. 45 illustrates primer sequence to clone canine NGF, SEQ ID NO: 45(NGF-Dog-S primer).

FIG. 46 illustrates primer sequence to clone canine NGF, SEQ ID NO: 46(NGF-Dog-AS primer).

FIG. 47 illustrates primer sequence to clone canine NGF, SEQ ID NO: 47(NGF-d-Ec-S primer).

FIG. 48 illustrates primer sequence to clone canine NGF, SEQ ID NO: 48(NGF-d-Ec-AS primer).

FIG. 49 illustrates canine NGF C-terminal 6His fusion nucleotidesequence, SEQ ID NO: 49.

FIG. 50 illustrates canine NGF C-terminal 6-His amino acid sequence, SEQID NO: 50.

FIG. 51 illustrates canine IgG constant region nucleotide sequence, SEQID NO: 51.

FIG. 52 illustrates canine IgG constant region amino acid sequence, SEQID NO: 52.

FIG. 53 illustrates canine kappa constant region nucleotide sequence,SEQ ID NO: 53

FIG. 54 illustrates canine kappa constant region amino acid sequence,SEQ ID NO: 54.

FIG. 55 illustrates complementarity determining region, SEQ ID NO: 55(72.1 VH amino acid; CDR1).

FIG. 56 illustrates complementarity determining region, SEQ ID NO: 56(72.1 VH amino acid; CDR2).

FIG. 57 illustrates complementarity determining region, SEQ ID NO: 57(72.1 VH amino acid; CDR3).

FIG. 58 illustrates complementarity determining region, SEQ ID NO: 58(72.1 VL amino acid; CDR1).

FIG. 59 illustrates complementarity determining region, SEQ ID NO: 59(72.1 VL amino acid; CDR2).

FIG. 60 illustrates complementarity determining region, SEQ ID NO: 60(72.1 VL amino acid; CDR3).

FIG. 61 illustrates complementarity determining region, SEQ ID NO: 61(73.1 VH amino acid; CDR1).

FIG. 62 illustrates complementarity determining region, SEQ ID NO: 62(73.1 VH amino acid; CDR2).

FIG. 63 illustrates complementarity determining region, SEQ ID NO: 63(73.1 VH amino acid; CDR3).

FIG. 64 illustrates complementarity determining region, SEQ ID NO: 64(73.1 VL amino acid; CDR1).

FIG. 65 illustrates complementarity determining region, SEQ ID NO: 65(73.1 VL amino acid; CDR2).

FIG. 66 illustrates complementarity determining region, SEQ ID NO: 66(73.1 VL amino acid; CDR3).

FIG. 67 illustrates complementarity determining region, SEQ ID NO: 67(77.1 VH amino acid; CDR1).

FIG. 68 illustrates complementarity determining region, SEQ ID NO: 68(77.1 VH amino acid; CDR2).

FIG. 69 illustrates complementarity determining region, SEQ ID NO: 69(77.1 VH amino acid; CDR3).

FIG. 70 illustrates complementarity determining region, SEQ ID NO: 70(77.1 VL amino acid; CDR1).

FIG. 71 illustrates complementarity determining region, SEQ ID NO: 71(77.1 VL amino acid; CDR2).

FIG. 72 illustrates complementarity determining region, SEQ ID NO: 72(77.1 VL amino acid; CDR3).

FIG. 73 illustrates complementarity determining region, SEQ ID NO: 73(81.1 VH amino acid; CDR1).

FIG. 74 illustrates complementarity determining region, SEQ ID NO: 74(81.1 VH amino acid; CDR2).

FIG. 75 illustrates complementarity determining region, SEQ ID NO: 75(81.1 VH amino acid; CDR3).

FIG. 76 illustrates complementarity determining region, SEQ ID NO: 76(81.1 VL amino acid; CDR1).

FIG. 77 illustrates complementarity determining region, SEQ ID NO: 77(81.1 VL amino acid; CDR2).

FIG. 78 illustrates complementarity determining region, SEQ ID NO: 78(81.1 VL amino acid; CDR3).

FIG. 79 illustrates complementarity determining region, SEQ ID NO: 79(82.1 VH amino acid; CDR1).

FIG. 80 illustrates complementarity determining region, SEQ ID NO: 80(82.1 VH amino acid; CDR2).

FIG. 81 illustrates complementarity determining region, SEQ ID NO: 81(82.1 VH amino acid; CDR3).

FIG. 82 illustrates complementarity determining region, SEQ ID NO: 82(82.1 VL amino acid; CDR1).

FIG. 83 illustrates complementarity determining region, SEQ ID NO: 83(82.1 VL amino acid; CDR2).

FIG. 84 illustrates complementarity determining region, SEQ ID NO: 84(82.1 VL amino acid; CDR3).

FIG. 85 illustrates the sequence of human βNGF (SEQ ID NO: 85).

FIG. 86 illustrates the sequences shown in Table 14 illustrating SEQ IDNOs 178, 86-88 from canine heavy chain variable domain sequences derivedfrom canine PBMC.

FIG. 86A illustrates the sequences shown in Table 14 illustrating SEQ IDNOs 89-93 from canine heavy chain variable domain sequences derived fromcanine PBMC.

FIG. 86B illustrates the sequences shown in Table 14 illustrating SEQ IDNOs 94-98 from canine heavy chain variable domain sequences derived fromcanine PBMC.

FIG. 86C illustrates the sequences shown in Table 14 illustrating SEQ IDNOs 99-102 from canine heavy chain variable domain sequences derivedfrom canine PBMC.

FIG. 86D illustrates the sequences shown in Table 14 illustrating SEQ IDNOs 103-107 from canine heavy chain variable domain sequences derivedfrom canine PBMC.

FIG. 86E illustrates the sequences shown in Table 14 illustrating SEQ IDNOs 108-109 from canine heavy chain variable domain sequences derivedfrom canine PBMC.

FIG. 87 illustrates the sequences shown in Table 15 illustrating SEQ IDNOs 110, 111, 204, 112 from canine lambda light chain variable domainsequences derived from canine PBMC RNA.

FIG. 87A illustrates the sequences shown in Table 15 illustrating SEQ IDNOs 113-117 from canine lambda light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 87B illustrates the sequences shown in Table 15 illustrating SEQ IDNOs 118-122 from canine lambda light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 87C illustrates the sequences shown in Table 15 illustrating SEQ IDNOs 123-126 from canine lambda light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 88 illustrates the sequences shown in Table 16 illustrating SEQ IDNOs 127-131 from canine kappa light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 88A illustrates the sequences shown in Table 16 illustrating SEQ IDNOs 132-136 from canine kappa light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 88B illustrates the sequences shown in Table 16 illustrating SEQ IDNOs 137-141 from canine kappa light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 88C illustrates the sequences shown in Table 16 illustrating SEQ IDNOs 142-146 from canine kappa light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 88D illustrates the sequences shown in Table 16 illustrating SEQ IDNOs 147-151 from canine kappa light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 88E illustrates the sequences shown in Table 16 illustrating SEQ IDNOs 152-156 from canine kappa light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 88F illustrates the sequences shown in Table 16 illustrating SEQ IDNOs 157-161 from canine kappa light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 88G illustrates the sequences shown in Table 16 illustrating SEQ IDNOs 162-164 from canine kappa light chain variable domain sequencesderived from canine PBMC RNA.

FIG. 89 illustrates the sequences shown in Table 17 illustrating SEQ IDNOs 165-168 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks(CDR-grafted Anti-NGF); CDRs underlined.

FIG. 89A illustrates the sequences shown in Table 17 illustrating SEQ IDNOs 169-173 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks(CDR-grafted Anti-NGF); CDRs underlined.

FIG. 89B illustrates the sequences shown in Table 17 illustrating SEQ IDNOs 174-176 from mouse anti-NGF CDRs grafted onto Human Ig Frameworks(CDR-grafted Anti-NGF); CDRs underlined.

FIG. 90 illustrates the sequences shown in Table 18 illustrating SEQ IDNOs 194-196 from Mouse/Canine Chimeric Antibody sequences.

FIG. 90A illustrates the sequences shown in Table 18 illustrating SEQ IDNOs 197-199 from Mouse/Canine Chimeric Antibody sequences.

FIG. 90B illustrates the sequences shown in Table 18 illustrating SEQ IDNOs 200-202 from Mouse/Canine Chimeric Antibody sequences.

FIG. 90C illustrates the sequences shown in Table 18 illustrating SEQ IDNOs 203 from Mouse/Canine Chimeric Antibody sequences.

DETAILED DESCRIPTION OF THE DISCLOSURE

The disclosure describes NGF binding proteins, particularly anti-NGFantibodies, or antigen-binding portions thereof, that bind NGF. Variousaspects of the disclosure relate to antibodies and antibody fragments,and pharmaceutical compositions thereof, as well as nucleic acids,recombinant expression vectors and host cells for making such antibodiesand fragments. Methods of using the antibodies of the disclosure todetect human and canine NGF, to inhibit human and canine NGF activity,either in vitro or in vivo; and to regulate gene expression are alsoencompassed by the disclosure.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well-known and commonly used in the art. Themethods and techniques of the present disclosure are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturers specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well-known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present disclosure may be more readily understood, select termsand phrases as used herein are defined below.

DEFINITIONS

The terms “acceptor” and “acceptor antibody” refer to the antibody ornucleic acid sequence providing or encoding at least 80%, at least 85%,at least 90%, at least 95%, at least 98% or 100% of the amino acidsequences of one or more of the framework regions. The term “acceptor”encompasses an antibody amino acid or nucleic acid sequence providing orencoding the constant region(s). The term also encompasses the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). For example, the term“acceptor” may refer to a human antibody amino acid or nucleic acidsequence that provides or encodes at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or 100% of the amino acid sequences ofone or more of the framework regions. Such an acceptor may contain atleast 1, at least 2, at least 3, least 4, at least 5, or at least 10amino acid residues that does (do) not occur at one or more specificpositions of a human antibody. An acceptor framework region and/oracceptor constant region(s) may be, e.g., derived or obtained from agermline antibody gene, a mature antibody gene, a functional antibody(e.g., antibodies well-known in the art, antibodies in development, orantibodies commercially available).

The term “agonist” refers to a modulator that, when contacted with amolecule of interest, causes an increase in the magnitude of a certainactivity or function of the molecule compared to the magnitude of theactivity or function observed in the absence of the agonist. Particularagonists of interest may include, but are not limited to, NGFpolypeptides or polypeptides, nucleic acids, carbohydrates, or any othermolecules that bind to NGF.

The term “antagonist” or “inhibitor” refer to a modulator that, whencontacted with a molecule of interest causes a decrease in the magnitudeof a certain activity or function of the molecule compared to themagnitude of the activity or function observed in the absence of theantagonist Particular antagonists of interest include those that blockor modulate the biological or immunological activity of NGF. Antagonistsand inhibitors of NGF may include, but are not limited to, proteins,nucleic acids, carbohydrates, or any other molecules, which bind to NGF.

The term “antibody” broadly refers to any immunoglobulin (Ig) moleculecomprised of four polypeptide chains, two heavy (H) chains and two light(L) chains, or any functional fragment, mutant, variant, or derivationthereof, which retains the essential epitope binding features of an Igmolecule. Such mutant, variant, or derivative antibody formats are knownin the art Non-limiting examples are discussed herein below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions may be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules may be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAI andIgA2) or subclass.

The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.In one aspect the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof.

The term “antibody construct” refers to a polypeptide comprising one ormore the antigen binding portions linked to a linker polypeptide or animmunoglobulin constant domain. Linker polypeptides comprise two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Such linker polypeptides are well knownin the art (Holliger, et al., Proc. Natl. Acad. Set, 90: 6444-6448(1993); Poljak, et al., Structure 2: 1121-1123 (1994)). Animmunoglobulin constant domain refers to a heavy or light chain constantdomain. Human IgG heavy chain and light chain constant domain amino acidsequences are known in the art; canine, equine, and feline are rarer.

The term “antibody fragments” or “antigen-binding moiety” comprises aportion of a full length antibody, generally the antigen binding orvariable domain thereof. Examples of antibody fragments include Fab,Fab′, F(ab)2, Fv, scFv fragments, diabodies, linear antibodies,single-chain antibody molecules.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”) refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., NGF). It has beenshown that the antigen-binding function of an antibody may be performedby fragments of a full-length antibody. These may also be bispecific,dual specific, or multi-specific formats; specifically binding to two ormore different antigens. Examples of binding fragments encompassedwithin the term “antigen-binding portion” of an antibody include (i) aFab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCTpublication WO 90/05144), which comprises a single variable domain; and(vi) an isolated complementarity determining region (CDR). Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they may be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv) (Bird et al., Science, 242: 423-426 (1988);and Huston et al., Proc. Natl. Acad. Sci., 85: 5879-5883 (1988)). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen-binding portion” of an antibody. Other forms of singlechain antibodies, such as diabodies are also encompassed.

Diabodies are bivalent, bispecific antibodies in which VH and VL domainsare expressed on a single polypeptide chain, but using a linker that istoo short to allow for pairing between the two domains on the samechain, thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (Holliger, et al.,Proc. Natl. Acad. Sci., 90: 6444-6448 (1993); Poljak, et al., Structure2: 1121-1123 (1994)). Such antibody binding portions are known in theart (Kontermann and Dubel eds., Antibody Engineering (2001)Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al., Human Antibodiesand Hybridomas, 6: 93-101 (1995)) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, et al., Mol. Immunol., 31:1047-1058 (1994)). Antibody portions, such as Fab and F(ab′)2 fragments,may be prepared from whole antibodies using conventional techniques,such as papain or pepsin digestion, respectively, of whole antibodies.Moreover, antibodies, antibody portions and immunoadhesion molecules maybe obtained using standard recombinant DNA techniques, as describedherein.

The term “anti-NGF antibody” refers to an antibody which is able to bindto nerve growth factor (NGF) and inhibit NGF biological activity and/ordownstream pathway(s) mediated by NGF signaling. An anti-NGF antibodyencompasses antibodies that block, antagonize, suppress or reduce(including significantly) NGF biological activity, including downstreampathways mediated by NGF signaling, such as receptor binding and/orelicitation of a cellular response to NGF. Anti-NGF antibodies encompassthose that neutralize NGF biological activity, bind NGF and prevent NGFdimerization and/or binding to an NGF receptor (such as p75 and/ortrkA), and/or bind NGF and prevent trkA receptor dimerization and/ortrkA autophosphorylation. Examples of anti-NGF antibodies are providedherein.

The term “binding protein” refers to a natural or synthetic polypeptidethat specifically binds to any portion of a target such as an antigen.The term “binding protein” encompasses antibodies as described herein,including an isolated antibody, antigen-binding portion thereof, orimmunologically functional fragment thereof

The term “canine antibody” refers to a naturally-occurring orrecombinantly produced immunoglobulin composed of amino acid sequencesrepresentative of natural antibodies isolated from canines of variousbreeds. Canine antibodies are antibodies having variable and constantregions derived from canine germline immunoglobulin sequences. Thecanine antibodies of the disclosure may include amino acid residues notencoded by canine germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “canine antibody” is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto canineframework sequences.

The term “caninization” is defined as a method for transferringnon-canine antigen-binding amino acids from a donor antibody to a canineantibody acceptor framework to generate protein therapeutic treatmentsuseful in dogs.

The term “caninized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-canine species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “canine-like”, i.e., more similarto canine germline variable sequences. One type of caninized antibody isa CDR-grafted antibody, in which non-canine CDR sequences are introducedinto canine VH and VL sequences to replace the corresponding canine CDRsequences.

Caninized forms of non-canine antibodies provided herein are canineantibodies that contain sequence derived from a non-canine antibody. Forthe most part, caninized antibodies are canine antibody sequences(“acceptor” or “recipient” antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-canine species (“donor” antibody) such as mouse, rat, rabbit,cat, goat, chicken, bovine, horse, llama, camel, dromedaries, sharks,non-human primates, human, humanized, recombinant sequence, or anengineered sequence having the desired properties. In some instances,framework region (FR) residues of the canine antibody are replaced bycorresponding non-canine FR residues. Furthermore, caninized antibodiesmay include residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. The caninized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a canineantibody. Strategies for caninization of antibodies include, but are notlimited to, the strategies disclosed in WO 2003/060080.

The caninized antibody is an antibody or a variant, derivative, analogor fragment thereof which immunospecifically binds to an antigen ofinterest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of a canine antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-canine antibody. A caninized antibody comprisessubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-canine immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of a canine immunoglobulin consensus sequence. A caninizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of a canine immunoglobulin. A canine orcaninized antibody may contain both the light chain as well as at leastthe variable domain of a heavy chain. The antibody also may include theCH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A caninizedantibody may only contain a caninized light chain, or may only contain acaninized heavy chain. An exemplary caninized antibody contains acaninized variable domain of a light chain and a caninized variabledomain of a heavy chain.

The term “canonical” residue refers to a residue in a CDR or frameworkthat defines a particular canonical CDR structure as defined by Chothiaet al. (J. Mol. Biol., 196:901-907 (1987); Chothia et al., J. Mol.Biol., 227:799 (1992). According to Chothia et al., critical portions ofthe CDRs of many antibodies have nearly identical peptide backboneconformations despite great diversity at the level of amino acidsequence. Each canonical structure specifies primarily a set of peptidebackbone torsion angles for a contiguous segment of amino acid residuesforming a loop.

The term “CDR” refers to the complementarity determining region withinantibody variable sequences. There are three CDRs in each of thevariable regions of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable regions. Theterm “CDR set” refers to a group of three CDRs that occur in a singlevariable region capable of binding the antigen. The exact boundaries ofthese CDRs have been defined differently according to different systems.The system described by Kabat (Kabat et al., Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987) and (1991)) not only provides an unambiguous residue numberingsystem applicable to any variable region of an antibody, but alsoprovides precise residue boundaries defining the three CDRs. These CDRsmay be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk,J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883(1989)) found that certain sub-portions within Kabat CDRs adopt nearlyidentical peptide backbone conformations, despite having great diversityat the level of amino acid sequence. These sub-portions were designatedas L11, L2 and L3 or H1, H2 and H3 where the “L” and the “H” designatesthe light chain and the heavy chains regions, respectively. Theseregions may be referred to as Chothia CDRs, which have boundaries thatoverlap with Kabat CDRs. Other boundaries defining CDRs overlapping withthe Kabat CDRs have been described by Padlan (FASEB J. 9: 133-139(1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDRboundary definitions may not strictly follow one of the above systems,but will nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, although certainmethods described herein use Kabat or Chothia defined CDRs.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human, canine, equine,or feline constant regions. Chimeric antibodies comprise a portion ofthe heavy and/or light chain that is identical to or homologous withcorresponding sequences from antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is identical to or homologous withcorresponding sequences in antibodies from another species or belongingto another antibody class or subclass, as well as fragments of suchantibodies, exhibiting the desired biological activity (See e.g., U.S.Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA81:6851-6855 (1984)).

The terms “crystal” and “crystallized” refer to an antibody, or antigenbinding portion thereof, that exists in the form of a crystal. Crystalsare one form of the solid state of matter, which is distinct from otherforms such as the amorphous solid state or the liquid crystalline state.Crystals are composed of regular, repeating, three-dimensional arrays ofatoms, ions, molecules (e.g., proteins such as antibodies), or molecularassemblies (e.g., antigen/antibody complexes). These three-dimensionalarrays are arranged according to specific mathematical relationshipsthat are well-understood in the field. The fundamental unit, or buildingblock, that is repeated in a crystal is called the asymmetric unit.Repetition of the asymmetric unit in an arrangement that conforms to agiven, well-defined crystallographic symmetry provides the “unit cell”of the crystal. Repetition of the unit cell by regular translations inall three dimensions provides the crystal. See Giege, R. and Ducruix, A.Barrett, Crystallization of Nucleic Acids and Proteins, a PracticalApproach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y.,(1999).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

The terms “donor” and “donor antibody” refer to an antibody providingone or more CDRs. A donor antibody may be an antibody from a speciesdifferent from the antibody from which the framework regions areobtained or derived. In the context of a humanized antibody, the term“donor antibody” refers to a non-human antibody providing one or moreCDRs. In the context of a caninized antibody, the term “donor antibody”refers to a non-canine antibody providing one or more CDRs. In thecontext of a felinized antibody, the term “donor antibody” refers to anon-feline antibody providing one or more CDRs. In the context of anequinized antibody, the term “donor antibody” refers to a non-equineantibody providing one or more CDRs.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopedeterminants may include chemically active surface groupings ofmolecules such as amino acids, sugar side chains, phosphoryl, orsulfonyl, and may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. An antibody is saidto specifically bind an antigen when it preferentially recognizes itstarget antigen in a complex mixture of proteins and/or macromolecules.

The term “equine antibody” refers to a naturally-occurring orrecombinantly produced immunoglobulin composed of amino acid sequencesrepresentative of natural antibodies isolated from equines of variousbreeds. Equine antibodies are antibodies having variable and constantregions derived from equine germline immunoglobulin sequences. Theequine antibodies of the disclosure may include amino acid residues notencoded by equine germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “equine antibody” is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto equineframework sequences.

The term “equalization” is defined as a method for transferringnon-equine antigen-binding amino acids from a donor antibody to anequine antibody acceptor framework to generate protein therapeutictreatments useful in horses.

The term “equinized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-equine species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “equine-like”, i.e., more similarto equine germline variable sequences. One type of equinized antibody isa CDR-grafted antibody, in which non-equine CDR sequences are introducedinto equine VH and VL sequences to replace the corresponding equine CDRsequences.

Equinized forms of non-equine antibodies provided herein are equineantibodies that contain sequence derived from a non-equine antibody. Forthe most part, equinized antibodies are equine antibody sequences(“acceptor” or “recipient” antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-equine species (“donor” antibody) such as mouse, rat, rabbit,cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries,sharks, non-human primates, human, humanized, recombinant sequence, oran engineered sequence having the desired properties. In some instances,framework region (FR) residues of the equine antibody are replaced bycorresponding non-equine FR residues. Furthermore, equinized antibodiesmay include residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. The equinized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of an equineantibody.

The equinized antibody is an antibody or a variant, derivative, analogor fragment thereof which immunospecifically binds to an antigen ofinterest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of an equine antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-equine antibody. An equinized antibody comprisessubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-equine immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of an equine immunoglobulin consensus sequence. An equinizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of an equine immunoglobulin. An equine orequinized antibody for example may contain both the light chain as wellas at least the variable domain of a heavy chain. The antibody also mayinclude the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Anequinized antibody may only contain an equinized light chain, or anequinized heavy chain. An exemplary equinized antibody contains anequinized variable domain of a light chain an equinized variable domainof a heavy chain. Equine isotypes include, for example, IgGa, IgGb,IgGc, IgG (T), IgM, and IgA

The term “Fab” refers to antibody fragments. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, whose name reflects its ability to readily crystallize.Pepsin treatment yields a binding cross-linking antigen. The Fabfragment also contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteine(s)from the antibody hinge region. Fab′-SH is the designation herein forFab′ in which the cysteine residue(s) of the constant domains bear afree thiol group. F(ab′)2 antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

The term “feline antibody” refers to a naturally-occurring orrecombinantly produced immunoglobulin composed of amino acid sequencesrepresentative of natural antibodies isolated from felines of variousbreeds. Feline antibodies are antibodies having variable and constantregions derived from feline germline immunoglobulin sequences. Thefeline antibodies of the disclosure may include amino acid residues notencoded by feline germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “feline antibody” is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto felineframework sequences.

The term “felinization” is defined as a method for transferringnon-feline antigen-binding amino acids from a donor antibody to a felineantibody acceptor framework to generate protein therapeutic treatmentsuseful in cats.

The term “felinized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-feline species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “feline-like”, i.e., more similarto feline germline variable sequences. One type of felinized antibody isa CDR-grafted antibody, in which non-feline CDR sequences are introducedinto feline VH and VL sequences to replace the corresponding feline CDRsequences.

Felinized forms of non-feline antibodies provided herein are felineantibodies that contain sequence derived from a non-feline antibody. Forthe most part, felinized antibodies are feline antibody sequences(“acceptor” or “recipient” antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-feline species (“donor” antibody) such as mouse, rat, rabbit,cat, dogs, goat, chicken, bovine, horse, llama, camel, dromedaries,sharks, non-human primates, human, humanized, recombinant sequence, oran engineered sequence having the desired properties. In some instances,framework region (FR) residues of the feline antibody are replaced bycorresponding non-feline FR residues. Furthermore, felinized antibodiesmay include residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. The felinized antibody may also comprise at leasta portion of an immunoglobulin constant region (Fc) of a felineantibody.

The felinized antibody is an antibody or a variant, derivative, analogor fragment thereof which immunospecifically binds to an antigen ofinterest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of a feline antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-feline antibody. A felinized antibody comprisessubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-feline immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of a feline immunoglobulin consensus sequence. A felinizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of a feline immunoglobulin. A feline orfelinized antibody may contain both the light chain as well as at leastthe variable domain of a heavy chain. The antibody also may include theCH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. A felinizedantibody may only contain a felinized light chain or a felinized heavychain. An exemplary felinized antibody only contains a felinizedvariable domain of a light chain and a felinized variable domain of aheavy chain.

The term “framework” or “framework sequence” refers to the remainingsequences of a variable region minus the CDRs. Because the exactdefinition of a CDR sequence may be determined by different systems, themeaning of a framework sequence is subject to correspondingly differentinterpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain andCDR-H1, -H2, and -H3 of heavy chain) also divide the framework regionson the light chain and the heavy chain into four sub-regions (FR1, FR2,FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 andFR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Withoutspecifying the particular sub-regions as FR1, FR2, FR3 or FR4, aframework region, as referred by others, represents the combined FR'swithin the variable region of a single, naturally occurringimmunoglobulin chain. An FR represents one of the four sub-regions, andFRs represents two or more of the four sub-regions constituting aframework region. Human heavy chain and light chain acceptor sequencesare known in the art. Canine heavy chain and light chain acceptorsequences are also known (patent application publication WO03/060080 andU.S. Pat. No. 7,261,890B2).

The term “germline antibody gene” or “gene fragment” refers to animmunoglobulin sequence encoded by non-lymphoid cells that have notundergone the maturation process that leads to genetic rearrangement andmutation for expression of a particular immunoglobulin (Shapiro et al.,Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al, Adv ExpMed Biol. 484: 13-30 (2001)). One of the advantages provided by thebinding proteins of the present disclosure stems from the recognitionthat germline antibody genes are more likely than mature antibody genesto conserve essential amino acid sequence structures characteristic ofindividuals in the species, hence less likely to be recognized as from aforeign source when used therapeutically in that species.

The term “Fv” refers to the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region consists of adimer of one heavy chain and one light chain variable domain.

The term “human antibody” refers to antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the disclosure may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody” is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which non-human CDR sequences are introducedinto human VH and VL sequences to replace the corresponding human CDRsequences.

The humanized antibody is an antibody or a variant, derivative, analogor fragment thereof which immunospecifically binds to an antigen ofinterest and which comprises a framework (FR) region havingsubstantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. A humanized antibody comprisessubstantially all, or at least one, and typically two, variable domains(Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of a human immunoglobulin consensus sequence. A humanizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. A humanized orcaninized antibody may contain both the light chain as well as at leastthe variable domain of a heavy chain. The antibody also may include theCH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Alternatively,a humanized antibody may only contain a humanized light chain, or ahumanized heavy chain. An exemplary humanized antibody contains ahumanized variable domain of a light chain and a humanized variabledomain of a heavy chain.

The bovanized, camelized, caninized, equinized, felinized, or humanizedantibody may be selected from any class of immunoglobulins, includingIgM, IgG, IgD, IgA and IgE, and any isotype, including withoutlimitation IgG1, IgG2, IgG3 and IgG4. The bovanized, camelized,caninized, equinized, felinized, or humanized antibody may comprisesequences from more than one class or isotype, and particular constantdomains may be selected to optimize desired effector functions usingtechniques well-known in the art.

The framework and CDR regions of a bovanized, camelized, caninized,equinized, felinized, or humanized antibody need not correspondprecisely to the parental sequences, e.g., the donor antibody CDR or theconsensus framework may be mutagenized by substitution, insertion and/ordeletion of at least one amino acid residue so that the CDR or frameworkresidue at that site does not correspond to either the donor antibody orthe consensus framework. Such mutations, however, will not be extensive.Usually, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% and at least 95% of the bovanized, camelized, caninized,equinized, felinized, or humanized antibody residues will correspond tothose of the parental FR and CDR sequences. The term “consensusframework” refers to the framework region in the consensusimmunoglobulin sequence. The term “consensus immunoglobulin sequence”refers to the sequence formed from the most frequently occurring aminoacids (or nucleotides) in a family of related immunoglobulin sequences(See e.g., Winnaker, From Genes to Clones (Veriagsgesellschaft,Weinheim, Germany 1987). In a family of immunoglobulins, each positionin the consensus sequence is occupied by the amino acid occurring mostfrequently at that position in the family. If two amino acids occurequally frequently, either may be included in the consensus sequence.

The term “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a “complementarity determiningregion” or “CDR” in the light chain variable domain and in the heavychain variable domain as defined by Kabat et al., 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991) and/or asdefined by (Chothia and Lesk, Mol. Biol. 196:901-917 (1987) and/or asdefined as “AbM loops” by Martin, et al., Proc. Natl. Acad. Sci. USA,86:9268-9272 (1989) and/or as defined by Lefranc et al., Nucleic AcidsRes, 27:209-212 (1999) in the international ImMunoGeneTics informationsystems database. “Framework” or “FR” residues are those variable domainresidues other than the hypervariable region residues as herein defined.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by comparing their sequences thereof, wherein “identity”refers more specifically to the degree of sequence relatedness betweennucleic acid molecules or polypeptides, as determined by the matchbetween strings of two or more nucleotide or two or more amino acidsequences. “Identity” measures the percent of identical matches betweenthe smaller of two or more sequences with gap alignments (if any)addressed by a particular mathematical model or computer program (i.e.,“algorithms”). The term “similarity” is used to refer to a relatedconcept with respect to the relationship of two or more nucleic acidmolecules or two or more polypeptide molecules. In contrast to“identity,” “similarity” refers to a measure of relatedness, whichincludes both identical matches and conservative substitution matches.For example, for two polypeptide sequences that have 50/100 identicalamino acids, and the remainder are all non-conservative substitutions,then the percent identity and similarity would both be 50%. With respectto the same two sequences, if 25 more positions had conservativesubstitutions, then the percent identity remains 50%, while percentsimilarity would be 75% (75/100). Identity and similarity of relatednucleic acids and polypeptides may be readily calculated by methods wellknown and readily available in the art, including but are not limitedto, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A. M.,ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICSAND GENOME PROJECTS, (Smith, D. W., ed.), 1993, Academic Press, NewYork; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin, A. M., andGriffin, H. G., eds.), 1994, Humana Press, New Jersey; von Heinje, G.,SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQUENCEANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991, M.Stockton Press, New York; Carillo et al., 1988, SIAM J. Applied Math.,48:1073; and Durbin et al., 1998, BIOLOGICAL SEQUENCE ANALYSIS,Cambridge University Press.

Preferred methods to determine identity are designed to provide thehighest match between the compared sequences, and are well described inreadily publicly available computer programs. Preferred suchcomputerized methods for determining identity between two sequencesinclude, but are not limited to, the GCG program package, including GAP(Devereux et al., 1984, Nucl Acid. Res., 12:387; Genetics ComputerGroup, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, andFASTA (Altschul et al., 1990, J. Mol Biol., 215:403-410). The BLASTXprogram is publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., 1990, supra). Thewell-known Smith Waterman algorithm may also be used to determineidentity.

The terms “individual,” “patient,” and “subject” are usedinterchangeably herein, to refer to mammals, including, but not limitedto, humans, murines, simians, felines, canines, equines, bovines,porcines, ovines, caprines, mammalian farm and agricultural animals,mammalian sport animals, and mammalian pets. Exemplary subjectscompanion animals, such as a dog, cat or horse.

An “isolated antibody” refers to an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds NGF is substantially free ofantibodies that specifically bind antigens other than NGF). An isolatedantibody that specifically binds NGF may, however, have cross-reactivityto other antigens, such as NGF molecules from other species. Moreover,an isolated antibody may be substantially free of other cellularmaterial and/or chemicals. The terms “isolated polynucleotide” and“isolated nucleic acid” as used interchangeably herein refer to apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin is not associated with all or aportion of a polynucleotide in which the isolated polynucleotide isfound in nature, or is linked to a another polynucleotide to which it isnot linked in nature, or is not found in nature within a largersequence. The term “isolated protein” or “isolated polypeptide” is aprotein or polypeptide that by virtue of its origin or source ofderivation is not associated with naturally associated components thataccompany it in its native state; is substantially free of otherproteins from the same species; is expressed by a cell from a differentspecies; or does not occur in nature. Thus, a polypeptide that ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A protein may also be renderedsubstantially free of naturally associated components by isolation,using protein purification techniques well known in the art.

The term “K_(d)” refers to the dissociation constant of a particularantibody-antigen interaction as is known in the art.

The term “K_(on)” is refers to the on rate constant for association ofan antibody to the antigen to form the antibody/antigen complex as isknown in the art.

The term “K_(off)” refers to the off rate constant for dissociation ofan antibody from the antibody/antigen complex as is known in the art.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al., Ann. NY Acad, Sci., 190:382-391(1971); and Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 (1991)). For the heavy chain variableregion, the hypervariable region ranges from amino acid positions 31 to35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acidpositions 95 to 102 for CDR3. For the light chain variable region, thehypervariable region ranges from amino acid positions 24 to 34 for CDR1,amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to97 for CDR3.

The term “key residue” refers to certain residues within the variableregion that have more impact on the binding specificity and/or affinityof an antibody, in particular a mammalized antibody such as humanized,caninized, equinized or felinized antibody. A key residue includes, butis not limited to, one or more of the following: a residue that isadjacent to a CDR, a potential glycosylation site (may be either N- orO-glycosylation site), a rare residue, a residue capable of interactingwith the antigen, a residue capable of interacting with a CDR, acanonical residue, a contact residue between heavy chain variable regionand light chain variable region, a residue within the Vernier zone, anda residue in the region that overlaps between the Chothia definition ofa variable heavy chain CDR1 and the Kabat definition of the first heavychain framework.

The term “labeled binding protein” refers to a protein with a labelincorporated that provides for the identification of the bindingprotein. In one aspect, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that may be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that may be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁵Ho, ¹⁵³Sm); fluorescent labels (e.g.,FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags); and magnetic agents, such as gadoliniumchelates.

The term “mammalization” refers to a method for transferring donorantigen-binding information to a mammalian antibody acceptor to generateuseful therapeutic treatments. More specifically, the invention providesmethods for felinization, equinization and caninization of antibodies.

The term “mammalized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a mammal species (e.g., amouse) but in which at least a portion of the VH and/or VL sequence hasbeen altered to be more like “mammal of interest,” see for example,humanized, caninized, equinized or felinized antibodies defined herein.Such mammalized antibodies include, but are not limited to, bovanized,camelized, caninized, equinized, felinized, or humanized antibodies.

The terms “modulate” and “regulate” are used interchangeably and referto a change or an alteration in the activity of a molecule of interest(e.g., the biological activity of NGF). Modulation may be an increase ora decrease in the magnitude of a certain activity or function of themolecule of interest. Exemplary activities and functions of a moleculeinclude, but are not limited to, binding characteristics, enzymaticactivity, cell receptor activation, and signal transduction.

The term “modulator” is a compound capable of changing or altering anactivity or function of a molecule of interest (e.g., the biologicalactivity of NGF). For example, a modulator may cause an increase ordecrease in the magnitude of a certain activity or function of amolecule compared to the magnitude of the activity or function observedin the absence of the modulator. A modulator may be an inhibitor, whichdecreases the magnitude of at least one activity or function of amolecule. Exemplary inhibitors include, but are not limited to,proteins, peptides, antibodies, peptibodies, carbohydrates or smallorganic molecules. Peptibodies are described, e.g., in WO01/83525.

The term “monoclonal antibody” refers to an antibody that is derivedfrom a single done, including any eukaryotic, prokaryotic, or phagedone; and not the method by which it is produced and is not limited toantibodies produced through hybridoma technology.

The term “multivalent binding protein” is used in this specification todenote a binding protein comprising two or more antigen binding sites.The multivalent binding protein is engineered to have the three or moreantigen binding sites, and is generally not a naturally occurringantibody. The term “multispecific binding protein” refers to a bindingprotein capable of binding two or more related or unrelated targets.Dual variable domain (DVD) binding proteins are binding proteins thatcomprise two or more antigen binding sites and are tetravalent ormultivalent binding proteins. Such DVDs may be monospecific, i.e.capable of binding one antigen or multispecific, i.e. capable of bindingtwo or more antigens. DVD binding proteins comprising two heavy chainDVD polypeptides and two light chain DVD polypeptides are referred to aDVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide,and a light chain DVD polypeptide, and two antigen binding sites. Eachbinding site comprises a heavy chain variable domain and a light chainvariable domain with a total of 6 CDRs involved in antigen binding perantigen binding site. DVD binding proteins and methods of making DVDbinding proteins are disclosed in U.S. patent application Ser. No.11/507,050 and incorporated herein by reference.

One aspect of the disclosure pertains to a DVD binding proteincomprising binding proteins capable of binding NGF. In another aspect,the DVD binding protein is capable of binding NGF and a second target.

The terms “nerve growth factor” and “NGF” refer to nerve growth factorand variants thereof that retain at least part of the biologicalactivity of NGF. NGF includes all mammalian species of native sequenceNGF, including murine, rat, human, rabbit, canine, feline, equine, orbovine.

TABLE 1 Sequence of NGF Protein Sequence Identifier Canine NGFC-terminal 6-His SEQ ID NO: 50 Human NGF SEQ ID NO: 85

The term “NGF receptor” refers to a polypeptide that is bound by oractivated by NGF. NGF receptors include the TrkA receptor and the p75receptor of any mammalian species, including, but are not limited to,human, canine, feline, equine, primate, or bovine.

The terms “NGF-related disease” and “NGF-related disorder” encompass anydisease or disorder in which the activity of NGF in a subject sufferingfrom the disease or disorder has been shown to be or is suspected ofbeing either responsible for the pathophysiology of the disease ordisorder, or a factor that contributes to a worsening of the disease ordisorder, which may occur as a result of increased levels of NGF orincreased sensitivity of the subject to NGF. Accordingly, an NGF-relateddisease or NGF-related disorder is a disease or disorder in whichreduction of NGF activity is expected to alleviate the symptoms and/orprogression of the disease or disorder. Such diseases and disorders maybe evidenced, for example, by an increase in the concentration of NGF ina biological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of NGF in serum, plasma, synovial fluid,etc. of the subject), which may be detected, for example, using ananti-NGF antibody as described above. Non-limiting examples of diseasesand disorders that may be treated with the antibodies of the disclosureinclude those diseases and disorders discussed in the section belowpertaining to pharmaceutical compositions of the antibodies of thedisclosure, and encompass acute pain resulting for example from surgeryor other trauma, and chronic pain.

The term “neutralizing” refers to neutralization of biological activityof a NGF when a binding protein specifically binds NGF. A neutralizingbinding protein is a neutralizing antibody, who's binding to NGF resultsin inhibition of a biological activity of NGF. The neutralizing bindingprotein binds NGF and reduces a biologically activity of NGF by at leastabout 20%, 40%, 60%, 80%, 85% or more. Inhibition of a biologicalactivity of NGF by a neutralizing binding protein may be assessed bymeasuring one or more indicators of NGF biological activity well knownin the art, including cell proliferation, cell morphology changes, cellsignaling, or any detectable cellular response resulting from binding ofNGF to the TrkA receptor.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” refers topolynucleotide sequences which are necessary to effect the expressionand processing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include components whose presence is essentialfor expression and processing, and may also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

The term “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion.

The term “polynucleotide” means a polymeric form of two or morenucleotides, either ribonucleotides or deoxynucleotides or a modifiedform of either type of nucleotide. The term includes single and doublestranded forms of DNA. The term “isolated polynucleotide” shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or somecombination thereof) that, by virtue of its origin, the “isolatedpolynucleotide” is not associated with all or a portion of apolynucleotide with which the “isolated polynucleotide” is found innature; is operably linked to a polynucleotide that it is not linked toin nature; or does not occur in nature as part of a larger sequence.

The term “polypeptide” refers to any polymeric chain of amino acids. Theterms “peptide” and “protein” are used interchangeably with the termpolypeptide and also refer to a polymeric chain of amino acids. The term“polypeptide” encompasses native or artificial proteins, proteinfragments and polypeptide analogs of a protein sequence. A polypeptidemay be monomelic or polymeric.

The term “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result.

The term “recombinant host cell” (or simply “host cell”) is intended torefer to a cell into which exogenous DNA has been introduced. It shouldbe understood that such terms are intended to refer not only to theparticular subject cell, but, to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell”. In one aspect, host cells include prokaryotic andeukaryotic cells selected from any of the Kingdoms of life. Eukaryoticcells include protist, fungal, plant and animal cells. In another aspecthost cells include, but are not limited to, the prokaryotic cell line E.coli; mammalian cell lines CHO, HEK 293 and COS; the insect cell lineSf9; and the fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

The term “recombinant antibody” refers to all species of antibodies orimmunoglobulins that are prepared, expressed, created or isolated byrecombinant means, such as antibodies expressed using a recombinantexpression vector transfected into a host cell, antibodies isolated froma recombinant, combinatorial human antibody library (Hoogenboom, TIBTech., 15: 62-70 (1997); Azzazy et al., Clin. Biochem., 35: 425-445(2002); Gavilondo et al., BioTechniques, 29: 128-145 (2002); Hoogenboomet al., Immunology Today, 21: 371-378 (2000)), antibodies isolated froman animal (e.g., a mouse) that is transgenic for human immunoglobulingenes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res.20:6287-6295; Kellermann et al., Current Opinion in Biotechnology, 13:593-597 (2002); Little et al., Immunology Today, 21: 364-370 (2000)) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of immunoglobulin gene sequences to other DNAsequences. Such recombinant antibodies have variable and constantregions derived from species-specific germline immunoglobulin sequences.Such recombinant antibodies may be subjected to in vitro mutagenesis(or, when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to species-specific germline VH and VL sequences, maynot naturally exist within the antibody germline repertoire in vivo.

The term “recovering” refers to the process of rendering a chemicalspecies such as a polypeptide substantially free of naturally associatedcomponents by isolation, e.g., using protein purification techniqueswell known in the art.

The term “sample” is used in its broadest sense. A “biological sample”includes, but is not limited to, any quantity of a substance from aliving thing or formerly living thing. Such living things include, butare not limited to, humans, mice, rats, monkeys, dogs, rabbits and otheranimals. Such substances include, but are not limited to, blood, serum,urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodesand spleen.

The term “single-chainFv” or “scFv” refers to antibody fragmentscomprising the VH and VL domains of antibody, wherein these domains arepresent in a single polypeptide chain. Generally, the Fv polypeptidefurther comprises a polypeptide linker between the VH and VL domainswhich enables the scFv to form the desired structure for antigenbinding. For a review of scFv, see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Veriag, New York, pp. 269-315 (1994).

The terms “specific binding” or “specifically binding” in reference tothe interaction of an antibody, a protein, or a peptide with a secondchemical species, mean that the interaction is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope) on the chemical species; for example, an antibody recognizesand binds to a specific protein structure rather than to proteinsgenerally. If an antibody is specific for epitope “A”, the presence of amolecule containing epitope A (or free, unlabeled A), in a reactioncontaining labeled “A” and the antibody, will reduce the amount oflabeled A bound to the antibody.

The term “substantially” in the context of a CDR refers to a CDR havingan amino acid sequence at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% identical to the amino acidsequence of a non-human antibody CDR.

The term “surface plasmon resonance” refers to an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore system (Pharmacia Biosensor AB,Uppsala, Sweden and Piscataway, N.J.). For further descriptions(Jonsson, et al. Ann. Biol. Clin. 51: 19-26 (1993); Jonsson, et al.,Biotechniques 11: 620-627 (1991); Johnsson, et al., J. Mol. Recognit. 8:125-131 (1995); and Johnnson, B., et al, Anal. Biochem., 198: 268-277(1991)).

The term “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount may bethe amount and/or duration of a therapy which is sufficient to reduce orameliorate the severity and/or duration of a disorder or one or moresymptoms thereof, prevent the advancement of a disorder, causeregression of a disorder, prevent the recurrence, development, onset orprogression of one or more symptoms associated with a disorder, detect adisorder, or enhance or improve the prophylactic or therapeuticeffect(s) of another therapy (e.g., prophylactic or therapeutic agent).A therapeutically effective amount of the antibody or antibody portionmay be determined by a person skilled in the art and may vary accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody, or antibody portion, are outweighed by the therapeuticallybeneficial effects.

The term “transformation” refers to any process by which exogenous DNAenters a host cell. Transformation may occur under natural or artificialconditions using various methods well known in the art. Transformationmay rely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. The method isselected based on the host cell being transformed and may include, butis not limited to, viral infection, electroporation, lipofection, andparticle bombardment. Such “transformed” cells include stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome. They also include cells which transiently express theinserted DNA or RNA for limited periods of time.

The term “transgenic organism” refers to an organism having cells thatcontain a transgene, wherein the transgene introduced into the organism(or an ancestor of the organism) expresses a polypeptide not naturallyexpressed in the organism. A “transgene” is a DNA construct, which isstably and operably integrated into the genome of a cell from which atransgenic organism develops, directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicorganism.

The term “vector” is intended to refer to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) may be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the disclosure is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “Vernier zone” refers to a subset of framework residues thatmay adjust CDR structure and fine-tune the fit to antigen as describedby Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

Anti NGF Binding Proteins

The present disclosure provides a novel family of binding proteins,murine antibodies, CDR grafted antibodies, mammalized (bovanized,camelized, caninized, equinized, felinized, or humanized) antibodies,and fragments thereof, capable of binding and modulating the biologicalactivity or function of NGF, including the capability of neutralizingNGF. The disclosure thus also provides a therapeutic means with which toinhibit NGF and provides compositions and methods for treating diseaseassociated with increased levels of NGF, particularly a disease,condition or disorder where increased levels of NGF, as compared to NGFlevels observed in comparable normal subjects, is detrimental.

Binding proteins of the present disclosure may be made by any of anumber of techniques known in the art and as described herein, includingculturing a host cell described herein in culture medium underconditions sufficient to produce a binding protein capable of bindingNGF.

Monoclonal antibodies may be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies may be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981).

Methods for producing and screening for specific antibodies usinghybridoma technology are well known in the art. Such methods include,for example, culturing a hybridoma cell secreting an antibody of thedisclosure wherein the hybridoma is generated by fusing splenocytesisolated from a mouse immunized with an antigen of the disclosure withmyeloma cells and then screening the hybridomas resulting from thefusion for hybridoma clones that secrete an antibody able to bind apolypeptide of the disclosure. Briefly, for example, mice may beimmunized with an NGF antigen. The NGF antigen may be administered, withor without an adjuvant, to stimulate the immune response. Such adjuvantsinclude complete or incomplete Freund's adjuvant, RIBI (muramyldipeptides) or ISCOM (immunostimulating complexes). Such adjuvants mayprotect the polypeptide from rapid dispersal by sequestering it in alocal deposit, or they may contain substances that stimulate the host tosecrete factors that are chemotactic for macrophages and othercomponents of the immune system. If a polypeptide is being administered,the immunization schedule will involve two or more administrations ofthe polypeptide, spread out over several weeks.

After immunization of an animal with an NGF antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-NGFantibody-containing serum may be obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-NGF antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen NGF are detected in the mouse serum, the mouse spleen may beharvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, such as, forexample, cells from cell line SP20 available from the ATCC. Hybridomasmay be selected and cloned by limited dilution. The hybridoma clones maythen be assayed by methods known in the art for cells that secreteantibodies capable of binding NGF. Ascites fluid, which generallycontains high levels of antibodies, may be generated by immunizing micewith positive hybridoma clones.

Antibody-producing immortalized hybridomas may be prepared from theimmunized animal. After immunization, the animal may be sacrificed andthe splenic B cells fused to immortalized myeloma cells as is well knownin the art (Harlow et al., supra). Alternatively, the myeloma cells maybe from a non-secretory cell line and do not secrete immunoglobulinpolypeptides. After fusion and antibiotic selection, the hybridomas maybe screened using NGF, or a portion thereof, or a cell expressing NGF.Initial screening may be performed, for example, using an enzyme-linkedimmunoassay (ELISA) or a radioimmunoassay (PJA). An example of ELISAscreening is provided in WO 00/37504.

Anti-NGF antibody-producing hybridomas may be selected, cloned andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

An exemplary animal system for preparing hybridomas is the mouse.Hybridoma production in the mouse is very well established, andimmunization protocols and techniques for isolation of immunizedsplenocytes for fusion are well known. Fusion partners (e.g., murinemyeloma cells) and fusion procedures are also known. Alternatively, thehybridomas may be produced in a non-human, non-mouse species such as arat, sheep, pig, goat, cattle or horse. Alternatively, human hybridomasmay be produced, in which a human non-secretory myeloma is fused with ahuman cell expressing an anti-NGF antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of thedisclosure may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

Recombinant antibodies may be generated from single, isolatedlymphocytes using a procedure referred to in the art as the selectedlymphocyte antibody method (SLAM), as described in U.S. Pat. No.5,627,052, PCT Publication WO 92/02551 and Babcock et al., Proc. Natl.Acad. Sci, 93: 7843-7848 (1996). In this method, single cells secretingantibodies of interest, e.g., lymphocytes derived from any one of theimmunized animals described in Section 1, are screened using anantigen-specific hemolytic plaque assay, wherein the antigen NGF, or afragment thereof, is coupled to sheep red blood cells using a linker,such as biotin, and used to identify single cells that secreteantibodies with specificity for NGF. Following identification ofantibody-secreting cells of interest, heavy- and light-chain variableregion cDNAs may be rescued from the cells by reverse transcriptase-PCRand these variable regions may then be expressed, in the context ofappropriate immunoglobulin constant regions (e.g., human constantregions), in mammalian host cells, such as COS or CHO cells. The hostcells transfected with the amplified immunoglobulin sequences, derivedfrom in vivo selected lymphocytes, may then undergo further analysis andselection in vitro, for example by panning the transfected cells toisolate cells expressing antibodies to NGF. The amplified immunoglobulinsequences further may be manipulated in vitro, such as by in vitroaffinity maturation methods such as those described in PCT PublicationWO 97/29131 and PCT Publication WO 00/56772.

Antibodies may be produced by immunizing a non-human animal comprisingsome or all of the human immunoglobulin loci with an NGF antigen. Forexample, human monoclonal antibodies directed against NGF may begenerated using transgenic mice carrying parts of the human immunesystem rather than the mouse system, referred to in the literature andherein as “HuMab” mice, contain a human immunoglobulin gene minilocusthat encodes unrearranged human heavy (μ and γ) and K light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg et al., 1994,Nature 368:856-859). These mice exhibit reduced expression of mouse IgMor κ and in response to immunization, and the introduced human heavychain and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgG K monoclonal antibodies.The preparation of HuMab mice is well described in the literature. (See,e.g., Lonberg et al., 1994, Nature 368:856-859; Lonberg, 1994, Handbookof Exp. Pharmacology 113:49-101; Taylor et al., 1994, InternationalImmunology 6:579-591; Lonberg & Huszar, 1995, Intern. Rev. Immunol.13:65-93; and Harding & Lonberg, 1995, Ann. N.Y. Acad. Sci 764:536-546).Alternatively, other known mouse strains such as the HCo7, HCo12, and KMtransgenic mice strains may be used to generate human anti-NGFantibodies.

Another suitable, though non-limiting example of a transgenic mouse isthe XENOMOUSE® transgenic mouse, which is an engineered mouse strainthat comprises large fragments of the human immunoglobulin loci and isdeficient in mouse antibody production. See, e.g., Green et al. NatureGenetics, 7: 13-21 (1994); and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364; WO91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00 09560, and WO00/037504. The XENOMOUSE® transgenic mouse produces an adult-like humanrepertoire of fully human antibodies, and generates antigen-specifichuman mAbs. The XENOMOUSE® transgenic mouse contains approximately 80%of the human antibody repertoire through introduction of megabase sized,germline configuration YAC fragments of the human heavy chain loci andlight chain loci (Mendez et al., Nature Genetics 15: 146-156 (1997),Green et al., J. Exp. Med., 188: 483-495 (1998)).

In vitro methods also may be used to make the antibodies of thedisclosure, wherein an antibody library is screened to identify anantibody having the desired binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, U.S. Pat. No. 5,223,409;PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;WO 93/01288; WO 92/01047; WO 92/09690; Fuchs et al. Bio/Technology, 9:1370-1372 (1991); Hay et al., Hum Antibody Hybridomas, 3: 81-85 (1992);Huse et al., Science, 246: 1275-1281 (1989); McCafferty et al., Nature,348: 552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993);Hawkins et al., J o/Biol, 226: 889-896 (1992); Clackson et al., Nature,352: 624-628 (1991); Gram et al., PNAS, 89: 3576-3580 (1992); Garrad etal., Bio/Technology, 9: 1373-1377 (1991); Hoogenboom et al., Nuc AcidRes, 19: 4133-4137 (1991); and Barbas et al., PNAS, 88: 7978-7982(1991), US patent application publication 20030186374, and PCTPublication No. WO 97/29131.

The recombinant antibody library may be from a subject immunized withNGF, or a portion of NGF. Alternatively, the recombinant antibodylibrary may be from a naive subject that has not been immunized withNGF, such as a canine antibody library from a canine subject that hasnot been immunized with canine NGF. Antibodies of the disclosure areselected by screening the recombinant antibody library with the peptidecomprising canine NGF to thereby select those antibodies that recognizeNGF. Methods for conducting such screening and selection are well knownin the art, such as described in the references in the precedingparagraph. To select antibodies of the disclosure having particularbinding affinities for hNGF, such as those that dissociate from canineNGF with a particular k_(off) rate constant, the art-known method ofsurface plasmon resonance may be used to select antibodies having thedesired k_(off) rate constant. To select antibodies of the disclosurehaving a particular neutralizing activity for hNGF, such as those with aparticular an IC₅o, standard methods known in the art for assessing theinhibition of hNGF activity may be used.

For example, the antibodies of the present disclosure may also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular aspect, such phage may be utilized todisplay antigen-binding domains expressed from a repertoire orcombinatorial antibody library (e. g., canine, human or murine). Phageexpressing an antigen binding domain that binds the antigen of interestmay be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and MI 3binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatmay be used to make the antibodies of the present disclosure includethose disclosed in Brinkman et al., J. Immunol. Methods, 182: 41-50(1995); Ames et al., J. Immunol. Methods, 184: 177-186 (1995);Kettleborough et al., Eur. J. Immunol, 24:952-958 (1994); Persic et al.,Gene, 187: 9-18 (1997); Burton et al., Advances in Immunology, 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage may be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments may also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869(1992); and Sawai et al., AJPJ 34:26-34 (1995); and Better et al.,Science, 240: 1041-1043 (1988) (said references incorporated byreference in their entireties).

Examples of techniques which may be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shuet al., PNAS, 90:7995-7999 (1993); and Skerra et al., Science, 240:1038-1040 (1988).

Alternatives to screening of recombinant antibody libraries by phagedisplay are known and include other methodologies for screening largecombinatorial libraries which may be applied to the identification ofdual specificity antibodies of the disclosure. One type of alternativeexpression system is one in which the recombinant antibody library isexpressed as RNA-protein fusions, as described in PCT Publication No. WO98/31700, and in Roberts et al., Proc. Natl. Acad. Sci., 94: 12297-12302(1997). In this system, a covalent fusion is created between a mRNA andthe peptide or protein that it encodes by in vitro translation ofsynthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, attheir 3′ end. Thus, a specific mRNA may be enriched from a complexmixture of mRNAs (e.g., a combinatorial library) based on the propertiesof the encoded peptide or protein, e.g., antibody, or portion thereof,such as binding of the antibody, or portion thereof, to the dualspecificity antigen. Nucleic acid sequences encoding antibodies, orportions thereof, recovered from screening of such libraries may beexpressed by recombinant means as described above (e.g., in mammalianhost cells) and, moreover, may be subjected to further affinitymaturation by either additional rounds of screening of mRNA-peptidefusions in which mutations have been introduced into the originallyselected sequence(s), or by other methods for affinity maturation invitro of recombinant antibodies, as described above.

In another approach the antibodies of the present disclosure may also begenerated or affinity matured using yeast display methods known in theart. In yeast display methods, genetic methods are used to tetherantibody domains to the yeast cell wall and display them on the surfaceof yeast. In particular, such yeast may be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e. g., human or murine). Examples of yeast displaymethods that may be used to make the antibodies of the presentdisclosure include those disclosed Wittrup, et al. U.S. Pat. No.6,699,658 incorporated herein by reference.

The antibodies or antigen binding fragments described herein may also beproduced by genetic engineering. For example, the technology forexpression of both heavy and light chain genes in E. coli is the subjectof the PCT patent applications: publication number WO 901443, WO901443,and WO 9014424 and in Huse et al., 1989 Science 246: 1275-81. Thepresent disclosure thus also encompasses the isolated nucleic acidsencoding any of the binding proteins described herein, as well as arecombinant vector comprising such a nucleic acid molecule, and a hostcell comprising such a recombinant vector.

A vector is a nucleic acid molecule, which may be a construct, capableof transporting another nucleic acid to which it has been linked. Avector may include any preferred or required operational elements.Preferred vectors are those for which the restriction sites have beendescribed and which contain the operational elements needed fortranscription of the nucleic acid sequence. Such operational elementsinclude for example at least one suitable promoter, at least oneoperator, at least one leader sequence, at least one terminator codon,and any other DNA sequences necessary or preferred for appropriatetranscription and subsequent translation of the nucleic acid sequence.Such vectors contain at least one origin of replication recognized bythe host organism along with at least one selectable marker and at leastone promoter sequence capable of initiating transcription of the nucleicacid sequence. A vector may be a plasmid into which additional DNAsegments may be ligated. A vector may be a viral vector, whereinadditional DNA segments may be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) may be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as a plasmid is the most commonlyused form of vector. However, the present disclosure is intended toinclude such other forms of expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions. By way ofexample and not limitation, suitable vectors include pcDNA, pTT(Durocher et al., Nucleic Acids Research, Vol 30, No. 2 (2002)); pTT3(pTT with additional multiple cloning site, pEFBOS (Mizushima et al.,Nucleic acids Research, Vol 18, No. 17 (1990)), pBV, pJV, pBJ, or pHybE(patent publication no.: US 2009/0239259 AI).

Sequences that are operably linked are in a relationship permitting themto function in their intended manner. A control sequence operably linkedto a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. Operably linked sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. Expression control sequences are polynucleotide sequencesthat are necessary to effect the expression and processing of codingsequences to which they are ligated. Expression control sequencesinclude appropriate transcription initiation, termination, promoter andenhancer sequences; efficient RNA processing signals such as splicingand polyadenylation signals; sequences that stabilize cytoplasmic mRNA;sequences that enhance translation efficiency (i.e., Kozak consensussequence); sequences that enhance protein stability; and when desired,sequences that enhance protein secretion. The nature of such controlsequences differs depending upon the host organism; in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence; in eukaryotes, such controlsequences generally include promoters and transcription terminationsequence. Control sequences may include components whose presence isessential for expression and processing, and may also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

A host cell may be transformed with a vector that introduces exogenousDNA into a host cell. Transformation may occur under natural orartificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, calcium-phosphate precipitation, DEAE-dextran transfection,particle bombardment and the like. Transformed cells include stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, and cells which transiently express the inserted DNA or RNAfor limited periods of time.

Host organisms such as host cells are cultured under conditionsappropriate for amplification of the vector and expression of theprotein, as well known in the art. Expressed recombinant proteins may bedetected by any of a number of methods also well known in the art.

Suitable host organisms include for example a prokaryotic or eukaryoticcell system. A eukaryotic cell may be a protist cell, animal cell, plantcell or fungal cell. A eukaryotic cell is for example an animal cellwhich may be a mammalian cell, avian cell, or an insect cell such as aninsect Sf9 cell. Cells from established and readily available may beused, such as but not limited to HeLa, MRC-5 or CV-1. The host cell maybe an E. coli cell or a yeast cell such as but not limited toSaccharomyces cerevisiae. Mammalian host cells for expressing therecombinant antibodies of the disclosure also include Chinese HamsterOvary (CHO cells) (including dhfr-CHO cells, described in Urlaub et al.,Proc. Natl. Acad. Sci., 77: 4216-4220 (1980), used with a DHFRselectable marker, e.g., as described in Kaufman et al., Mol. Biol, 159:601-621 (1982)), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells, or by secretion of the antibody into theculture medium in which the host cells are grown. Antibodies may berecovered from the culture medium using standard protein purificationmethods.

Host cells may also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentdisclosure. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this disclosure. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of thedisclosure. In addition, bifunctional antibodies may be produced inwhich one heavy and one light chain are an antibody of the disclosureand the other heavy and light chain are specific for an antigen otherthan the antigens of interest by crosslinking an antibody of thedisclosure to a second antibody by standard chemical crosslinkingmethods.

In a system for recombinant expression of an antibody, orantigen-binding portion thereof, of the disclosure, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium.

Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium. Still further the disclosure provides a method ofsynthesizing a recombinant antibody of the disclosure by culturing ahost cell of the disclosure in a suitable culture medium until arecombinant antibody of the disclosure is synthesized. The method mayfurther comprise isolating the recombinant antibody from the culturemedium.

The present disclosure thus provides anti NGF binding proteins that arespecific for and substantially neutralize NGF polypeptides, includingactive human NGF. Also provided are antibody heavy and light chain aminoacid sequences which are substantially specific for and substantiallyneutralize NGF polypeptides when they are bound to them. Thisspecificity enables the anti-human NGF human antibodies and humanmonoclonal antibodies with like specificity, to be effectiveimmunotherapy for NGF associated diseases.

The present disclosure encompasses anti NGF binding proteins comprisingat least one of the amino acid sequences selected from the groupconsisting of: SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14,SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ IDNO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 165, SEQ ID NO: 166,SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ IDNO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184,SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ IDNO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201,SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 207 and SEQ ID NO: 4, SEQ IDNO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ IDNO: 44, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174,SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ IDNO: 186, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194,SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, andwhich binds an NGF polypeptide epitope with substantially high affinityas described herein and has the capacity to substantially modulate,including substantially reduce, NGF polypeptide activity.

Examples of such binding proteins include binding proteins comprising avariable heavy chain polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ IDNO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO:182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206 and SEQ ID NO: 207;and a variable light chain polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ IDNO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ IDNO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175,SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ IDNO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196,SEQ ID NO: 198, SEQ ID NO: 200 and SEQ ID NO: 202.

Exemplary pairings of a variable heavy chain polypeptide and a variablelight chain polypeptide are represented by the following pairings: SEQID NO: 2 and SEQ ID NO: 4; SEQ ID NO: 6 and SEQ ID NO: 8; SEQ ID NO: 10and SEQ ID NO: 12; SEQ ID NO: 14 and SEQ ID NO: 16; SEQ ID NO: 18 andSEQ ID NO: 20; SEQ ID NO: 22 and SEQ ID NO: 24; SEQ ID NO: 25 and SEQ IDNO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO:30; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 177 and SEQ ID NO: 32;SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO:36; SEQ IDNO: 37 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41and SEQ ID NO: 42; SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 180 andSEQ ID NO: 181, SEQ ID NO: 182 and SEQ ID NO: 183; SEQ ID NO: 185 andSEQ ID NO: 186; SEQ ID NO: 187 and SEQ ID NO: 188; and SEQ ID NO: 192and SEQ ID NO: 193.

Also encompassed in the disclosure are binding proteins thatspecifically bind NGF as described herein and comprise a heavy chainvariable region having at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with any of SEQ ID NO: 2, SEQ ID NO:6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO:169, SEQ ID NO: 170, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 180, SEQID NO: 182, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO:189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 197, SEQID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO:207. Also encompassed are binding proteins that specifically bind NGF asdescribed herein and comprise a light chain variable region having atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with any of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ IDNO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, SEQ ID NO: 171, SEQ IDNO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176,SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 188, SEQ IDNO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198,SEQ ID NO: 200, SEQ ID NO: 202.

Exemplary binding proteins that specifically bind NGF as describedherein preferably comprise a heavy chain variable region and a lightchain variable region as follows:

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 2, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and thelight chain variable region comprising an amino acid sequence having atleast 90% identity with SEQ ID NO: 4 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof; a heavychain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 6 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 8 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 10 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 12 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 14 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 16 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof; a heavychain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 18 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 20 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 22 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 24 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 25 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 26 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 27 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 28 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 29 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 30 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 31 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 32 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 177 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 32 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 33 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 34 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 35 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 36 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 37 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 38 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 39 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 40 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 41 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 42 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 43 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 44 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 180 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 181 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 182 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 183 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 185 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 186 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 187 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 188 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 189 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 42 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 190 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 188 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 206 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 42 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof;

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 207 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 188 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof; and

a heavy chain variable region comprising an amino acid sequence havingat least 90% identity with SEQ ID NO: 192 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof, and a lightchain variable region comprising an amino acid sequence having at least90% identity with SEQ ID NO: 193 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

Exemplary binding proteins as disclosed herein may include at least oneCDR comprising an amino acid sequence selected from: a) heavy chain CDRsconsisting of SEQ ID NOS: 55, 56, 57, 61, 62, 63, 67, 68, 69, 73, 74,75, 79, 80, 81; or modified CDR amino acid sequences having a sequenceidentity of at least 50%, at least 60%, at least 70%, at least 80%, orat least 90% to one of said sequences; b) light chain CDRs consisting ofSEQ ID NOS: 58, 59, 60, 64, 65, 66, 70, 71, 72, 76, 77, 78, 82, 83, 84;or modified CDR amino acid sequences having a sequence identity of atleast 50% %, at least 60%, at least 70%, at least 80%, or at least 90%to one of said sequences.

It should be understood that variations are contemplated in any of thenucleic acid and amino acid sequences described herein. Such variationsinclude those that will result in a nucleic acid sequence that iscapable of directing production of analogs of the corresponding NGFbinding proteins. It will be understood that due to the degeneracy ofthe genetic code, many substitutions of nucleotides may be made thatwill lead to a DNA sequence that remains capable of directing productionof the corresponding protein or its analogs. All such variant DNAsequences that are functionally equivalent to any of the sequencesdescribed herein are encompassed by the present disclosure.

A variant of any of the binding proteins described herein means aprotein (or polypeptide) that differs from a given protein (e.g., ananti-NGF antibody) in amino acid sequence by the addition (e.g.,insertion), deletion, or conservative substitution of amino acids, butthat retains the biological activity of the given protein. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity and degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges may be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (see, e.g., Kyte et al., J.Mol. Biol. 157: 105-132 (1982)). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes may besubstituted and still retains protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids also may be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.4,554,101, which is incorporated herein by reference). Substitution ofamino acids having similar hydrophilicity values may result in peptidesretaining biological activity, for example immunogenicity, as isunderstood in the art. In one aspect, substitutions are performed withamino acids having hydrophilicity values within ±2 of each other. Boththe hydrophobicity index and the hydrophilicity value of amino acids areinfluenced by the particular side chain of that amino acid. Consistentwith that observation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. “Variant” also may be used to describe apolypeptide or fragment thereof that has been differentially processed,such as by proteolysis, phosphorylation, or other post-translationalmodification, yet retains its biological activity or antigen reactivity,e.g., the ability to bind to NGF. Use of “variant” herein is intended toencompass fragments of a variant unless otherwise contradicted bycontext.

The binding proteins described herein encompass an immunoglobulinmolecule, disulfide linked Fv, scFv, monoclonal antibody, murineantibody, chimeric antibody, single domain antibody, CDR-graftedantibody, diabody, mammalized (bovanized, camelized, caninized,equinized, felinized, or humanized) antibody, a canine antibody, felineantibody, equine antibody, murine antibody, multispecific antibody, Fab,dual specific antibody, DVD, Fab′, bispecific antibody, F(ab′)2, or Fvincluding a single chain Fv fragment.

A binding protein may comprise a particular heavy chain constant region,such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region.An exemplary binding protein includes an IgG1 heavy chain constantregion or an IgG4 heavy chain constant region. Furthermore, the antibodymay comprise a light chain constant region, such as a kappa light chainconstant region or a lambda light chain constant region. An exemplarybinding protein comprises a kappa light chain constant region.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (Winter, et al. U.S. Pat. Nos.5,648,260; 5,624,821). The Fc portion of an antibody mediates severalimportant effector functions e.g. cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC) andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcyRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.At least one amino acid residue may be replaced in the constant regionof the antibody, for example the Fc region of the antibody, such thateffector functions of the antibody are altered.

Binding proteins according to the present disclosure may comprise aheavy chain immunoglobulin constant domain such as, for example, a humanor canine or equine or feline IgM constant domain, a human or canine orequine or feline IgG4 constant domain, a human or canine or equine orfeline IgG1 constant domain, a human or canine or equine or feline IgEconstant domain, a human or canine or equine or feline IgG2 constantdomain, a human or canine or equine or feline IgG3 constant domain, anda human or canine or equine or feline IgA constant domain. A bindingprotein as described herein may comprise a light chain immunoglobulinconstant domain such as but not limited to any of human, canine, equineor feline, kappa or lambda constant domains, or any of canine, equine orfeline kappa or lambda equivalent constant domains. An exemplary suchbinding protein has a constant region having an amino acid sequence ofSEQ ID NO: 52 or SEQ ID NO: 54.

Binding proteins as described herein may also encompass an NGFanti-idiotype antibody relative to at least one NGF binding protein ofthe present disclosure. The anti-idiotype antibody includes any proteinor peptide containing molecule that comprises at least a portion of animmunoglobulin molecule such as, but not limited to, at least onecomplimentarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or; any portion thereof, which may be incorporated into abinding protein of the present disclosure.

The binding proteins of the disclosure are capable of binding to humanand canine NGF with high specificity, and additionally are capable ofmodulating the biological activity or function of NGF in an organism ora subject, including substantially neutralizing human and canine NGF.Also encompassed by the present disclosure are isolated murinemonoclonal antibodies, or antigen-binding portions thereof, that bind toNGF with a substantially high affinity, have a slow off rate and/or havea substantially high neutralizing capacity. An exemplary binding proteinas disclosed herein is capable of neutralizing NGF with a potency (IC₅o)of at least about 10 nM, at least about 5 nM, at least about 1 nM, atleast about 0.5 nM, at least about 0.1 nM, at least about 0.05 nM, atleast about 0.01 nM, or at least about 0.001 nM, as measured in the TF-1cell proliferation assay or the pERK and Pathhunter assays. Bindingproteins as described herein may have an on rate constant (K) to NGF ofat least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹s⁻¹; at least about10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; at least about 10⁶M⁻¹s⁻¹; or atleast about 10⁷M⁻¹s⁻¹ as measured by surface plasmon resonance. Bindingproteins as described herein may have an off rate constant (K_(off)) toNGF of at most about 10⁻³ s⁻¹; at most about 10⁻⁴ s⁻¹; at most about10⁻⁵ s⁻¹; at most about 10⁻⁶ s⁻¹ or at most about 10⁻⁷ s⁻¹, as measuredby surface plasmon resonance. Binding proteins as described herein mayhave a dissociation constant (K_(D)) to NGF of at most about 10⁻⁷ M; atmost about 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰M; at mostabout 10⁻¹¹M; at most about 10⁻¹²M; at most about 10⁻¹³M, or at mostabout 10⁻¹⁴M. For example, a binding protein as described herein mayhave a dissociation constant (K_(D)) of about 1×10⁻⁹M, about 1×10⁻¹⁰ M,about 3.14×10⁻¹⁰M, about 1×10⁻¹¹M, about 2.37×10⁻¹¹M, about 1×10⁻¹²Mabout 1×10⁻¹³M or about 3.3×10⁻¹⁴M.

Binding proteins as described herein including an isolated antibody, orantigen-binding portion thereof, or immunologically functional fragmentthereof, may bind NGF and dissociate from NGF with a k_(off) rateconstant of about 0.1 s⁻¹ or less, as determined by surface plasmonresonance, or may inhibit NGF activity with an IC₅₀ of about 1×10⁻⁶M orless. Alternatively, the antibody, or an antigen-binding portionthereof, may dissociate from NGF with a k_(off) rate constant of about1×10⁻² s⁻¹ less, as determined by surface plasmon resonance, or mayinhibit NGF activity with an IC₅₀ of about 1×10⁻⁷ M or less.Alternatively, the antibody, or an antigen-binding portion thereof, maydissociate from NGF with a k_(off) rate constant of about 1×10³ s⁻¹ orless, as determined by surface plasmon resonance, or may inhibit NGFwith an IC₅0 of about 1×10⁻⁸M or less. Alternatively, the antibody, oran antigen-binding portion thereof, may dissociate from NGF with ak_(off) rate constant of about 1×10⁻⁴ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit NGF activity with an IC₅₀ ofabout 1×10⁻⁹M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from NGF with a k_(off)rate constant of about 1×10⁻⁵ s⁻¹ or less, as determined by surfaceplasmon resonance, or may inhibit NGF activity with an IC₅0 of about1×10⁻¹⁰M or less. Alternatively, the antibody, or an antigen-bindingportion thereof, may dissociate from NGF with a k_(off) rate constant ofabout 1×10⁻⁵ s⁻¹ less, as determined by surface plasmon resonance, ormay inhibit NGF activity with an IC₅₀ of about 1×10⁻¹¹M or less.

A binding protein as described herein may bind canine NGF, wherein theantibody, or antigen-binding portion thereof, may dissociate from canineNGF with a k_(off) rate constant of about 0.1 s⁻¹ or less, as determinedby surface plasmon resonance, or may inhibit canine NGF activity with anIC₅0 of about 1×10⁻⁶M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from canine NGF with ak_(off) rate constant of about 1×10⁻² s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit canine NGF activity with anIC₅0 of about 1×10⁻⁷M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from canine NGF with ak_(off) rate constant of about 1×10⁻³ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit canine NGF with an IC₅o ofabout 1×10⁻⁸M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from canine NGF with ak_(off) rate constant of about 1×10⁻⁴ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit canine NGF activity with anIC₅₀ of about 1×10⁻¹⁰M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from canine NGF with ak_(off) rate constant of about 1×10⁻⁵ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit canine NGF activity with anIC₅₀ of about 1×10⁻¹⁰M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from canine NGF with ak_(off) rate constant of about 1×10⁻⁵ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit canine NGF activity with anIC₅₀ of about 1×10⁻¹¹M or less.

The binding proteins of the disclosure further encompass bindingproteins coupled to an immunoadhesion molecule, imaging agent,therapeutic agent, or cytotoxic agent. Non-limiting examples of suitableimaging agents include an enzyme, fluorescent label, luminescent label,bioluminescent label, magnetic label, biotin or a radiolabel including,but not limited to, 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, ¹⁷⁷Lu,166Ho, and 153Sm. The therapeutic or cytotoxic agent may be ananti-metabolite, alkylating agent, antibiotic, growth factor, cytokine,anti-angiogenic agent, anti-mitotic agent, anthracydine, toxin, orapoptotic agent. Also provided herein is a labeled binding proteinwherein an antibody or antibody portion of the disclosure is derivatizedor linked to another functional molecule (e.g., another peptide orprotein). For example, a labeled binding protein of the disclosure maybe derived by functionally linking an antibody or antibody portion ofthe disclosed binding protein (by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody (e.g., a bispecific antibody or adiabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent,and/or a protein or peptide that may mediate associate of the antibodyor antibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

Useful detectable agents with which an antibody or antibody portion ofthe disclosure may be derivatized, may include fluorescent compounds.Exemplary fluorescent detectable agents include, for example,fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and thelike. An antibody may also be derivatized with detectable enzymes, suchas alkaline phosphatase, horseradish peroxidase, glucose oxidase and thelike. When an antibody is derivatized with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce adetectable reaction product. For example, when the detectable agenthorseradish peroxidase is present, addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

The binding proteins described herein may be in crystallized form.Crystallized binding proteins according to the present disclosure may beproduced according to methods known in the art, as disclosed for examplein WO 02072636. Preferably the crystallized binding protein retainsbiological activity after crystallization. The binding proteins may thusbe provided as crystals of whole anti-NGF antibodies or portions orfragments thereof as disclosed herein. Such crystals may be used toprepare formulations and compositions incorporating anti NGF bindingproteins, including diagnostic and therapeutic compositions. Anexemplary such crystallized binding protein is a carrier-free,controlled release crystallized binding protein. An exemplarycrystallized binding protein demonstrates a greater half-life in vivothan the soluble counterpart of the binding protein.

Anti NGF binding proteins as described herein may be glycosylated. Theglycosylation may demonstrate, for example, a bovine, camel, canine,murine, equine, feline, or human glycosylation pattern. Glycosylatedbinding proteins as described herein include the antibody orantigen-binding portion coupled to one or more carbohydrate residues.Nascent in vivo protein production may undergo further processing, knownas post-translational modification. Sugar (glycosyl) residues may beadded enzymatically, a process known as glycosylation. The resultingproteins bearing covalently linked oligosaccharide side chains are knownas glycosylated proteins or glycoproteins. Protein glycosylation dependson the amino acid sequence of the protein of interest, as well as thehost cell in which the protein is expressed. Different organisms mayproduce different glycosylation enzymes (ex., glycosyltransferases andglycosidases), and have different substrates (nucleotide sugars)available. Due to such factors, protein glycosylation pattern, andcomposition of glycosyl residues, may differ depending on the hostsystem in which the particular protein is expressed. Glycosyl residuesuseful in the disclosure may include, but are not limited to, glucose,galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Theglycosylated binding protein comprises glycosyl residues such that theglycosylation pattern is human, murine, canine, feline, bovine orequine.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the hostendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, such as a glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art, apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S patent applications20040018590 and 20020137134).

Further, it will be appreciated by those skilled in the art that aprotein of interest may be expressed using a library of host cellsgenetically engineered to express various glycosylation enzymes suchthat member host cells of the library produce the protein of interestwith variant glycosylation patterns. A practitioner may then select andisolate the protein of interest with particular novel glycosylationpatterns. The protein having a particularly selected novel glycosylationpattern exhibits improved or altered biological properties.

Anti NGF Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and anon-murine immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art, see e.g., Morrison, Science,229: 1202 (1985); Oi et al., BioTechniques, 4: 214 (1986); Gillies etal., J. Immunol. Methods, 125: 191-202 (1989); U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397. In addition, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad.Sci. 81: 851-855 (1984); Neuberger et al., Nature, 312:604-608 (1984);Takeda et al., Nature, 314: 452-454 (1985)) by splicing genes from amouse antibody molecule of appropriate antigen specificity together withgenes from a human antibody molecule of appropriate biological activitymay be used.

Anti NGF CDR Grafted Antibodies

CDR-grafted antibodies of the disclosure may comprise heavy and lightchain variable region sequences from a non-murine antibody wherein oneor more of the CDR regions of V_(H) and/or V_(L) are replaced with CDRsequences of the murine antibodies of the disclosure. A frameworksequence from any non-murine antibody may serve as the template for CDRgrafting. However, straight chain replacement onto such a frameworkoften leads to some loss of binding affinity to the antigen. The morehomologous a non-murine antibody is to the original murine antibody, theless likely the possibility that combining the murine CDRs with thenon-murine framework will introduce distortions in the CDRs that couldreduce affinity.

A non-murine variable framework that is chosen to replace the murinevariable framework apart from the CDRs may have at least 50%, at least60%, at least 70%, at least 80% or at least 90% sequence identity withthe murine antibody variable region framework. The non-murine variableframework, apart from the CDRs, that is chosen to replace the murinevariable framework, apart from the CDRs, may be a bovine, camel, canine,equine, feline or human variable framework. For example, the non-murinevariable framework that is chosen to replace the murine variableframework, apart from the CDRs, is a canine variable framework and hasat least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identitywith the murine antibody variable region framework.

Methods for producing CDR-grafted antibodies are known in the art (seeEP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089), and include veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5): 489-498(1991); Studnicka et al., Protein Engineering, 7(6):805-814 (1994);Roguska et al., PNAS, 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,352).

Anti NGF Humanized Antibodies

The process of modifying a monoclonal antibody from an animal to renderit less immunogenic for therapeutic administration to humans(humanization) has been aggressively pursued and has been described in anumber of publications (Antibody Engineering: A practical Guide. Carl A.K. Borrebaeck ed. W.H. Freeman and Company, 1992; and references citedabove). Humanized antibodies are antibody molecules from non-humanspecies antibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed in a variety of websites which are availableon the Internet (such as the NCBI website, Antibody Resource, and knownto those skilled in the art as well as in Kabat et al., Sequences ofProteins of Immunological Interest, U.S. Dept. Health (1983), which isincorporated herein by reference. Additional sequences are shown inTable 1 A below. Such imported sequences may be used to reduceimmunogenicity or reduce, enhance or modify binding, affinity, on-rate,off-rate, avidity, specificity, half-life, or any other suitablecharacteristic of the antibody, as known in the art.

TABLE 1A Mouse Anti-NGF mAb CDRs Grafted onto Human Ig Frameworks CDR-Grafted Anti-NGF Abs (ThisTable 1A is identical to Table 17 in the Examples) HU72 VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYM (CDR GRAFTFWVRQATGKGLEWVSTISDGGSYTYYTDNVKGRF VH3-13/JH5)TISRENAKNSLYLQMNSLRAGDTAVYYCARDWSD SEGFAYWGQGTLVTVSS (SEQ ID NO: 165)Hu73 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWM (CDR GRAFTHWVRQAPGQGLEWMGRIDPYGGGTKHNEKFKRRV VH1-18/JH6)TMTTDTSTSTAYMELRSLRSDDTAVYYCARSGYD YYFDVWGQGTTVTVSS (SEQ ID NO: 166)HU77 VH QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYI (CDR GRAFTYWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRV VH1-69/JH6)TITADKSTSTAYMELSSLRSEDTAVYYCARYGYY AYWGQGTTVTVSS (SEQ ID NO: 167)HU80 VH QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYI (CDR GRAFTYWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRV VH1-18/JH6)TMTTDTSTSTAYMELRSLRSDDTAVYYCARYGYY AYWGQGTTVTVSS (SEQ ID NO: 168)HU81 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSNHYM (CDR GRAFTYWVRQAPGKGLEWVGSISDGGAYTFYPDTVKGRF VH3-15/JH1)TISRDDSKNTLYLQMNSLKTEDTAVYYCTTEESA NNGFAFWGQGTLVTVSS (SEQ ID NO: 169)HU82 VH QVTLKESGPVLVKPTETLTLTCTVSGFSLTGYNI (CDR GRAFTNWIRQPPGKALEWLAMIWGYGDTDYNSALKSRLT VH2-26/JH6)ISKDTSKSQVVLTMTNMDPVDTATYYCARDHYGG NDWYFDVWGQGTTVTVSS (SEQ ID NO: 170)HU72 VL DIVMTQTPLSLPVTPGEPASISCRSSQSIVQSNG (CDR GRAFTNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFS 01/JK2)GSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFT FGQGTKLEIKR (SEQ ID NO: 171) HU73 VLDIQMIQSPSFLSASVGDRVSIICRASENIYSFLA (CDR GRAFTWYLQKPGKSPKLFLYNANTLAEGVSSRFSGRGSG L22/JK2)TDFTLTIISLKPEDFAAYYCQHHFGTPFTFGQGT KLEIKR (SEQ ID NO: 172) HU77 VLDIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDG (CDR GRAFTFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFS 01/JK2)GSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTF GQGTKLEIKR (SEQ ID NO: 173) HU80 VLDIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDG (CDR GRAFTFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFS 01/JK2)GSGSGTDFTLKISRVEAEDVGVYYCFESNYLFTF GQGTKLEIKR (SEQ ID NO: 174) HU81 VLDIVMTQTPLSLPVTPGEPASISCRSSQSILHSNG (CDR GRAFTNTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFS 01/JK2)GSGSGTDFTLKISRVEAEDVGVYYCFQGAHVPFT FGQGTKLEIKR (SEQ ID NO: 175) HU82 VLDIQMTQSPSSLSASVGDRVTITCRASQDITNYLN (CDR GRAFTWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSG 08/JK2)TDFTFTISSLQPEDIATYYCQQGKTLPRTFGQGT KLEIKR (SEQ ID NO: 176)

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter orimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988),which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues may beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies may be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239: 1534 (1988)), Sims etal., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol.196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, MolecularImmunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; and 4,816,567.

Anti NGF Caninized Antibodies

The process of modifying a monoclonal antibody from an animal to renderit less immunogenic for therapeutic administration to canines(caninization) has been described in U.S. Pat. No. 7,261,890 B2 2007).The amino acid sequence of canine IgG1 is provided in GenBank(AF354264). Determination of the amino acid sequence of the variableregions of both a canine IgM and a canine IgA heavy chain (Wasserman etal., Biochem., 16, 3160 (1977), determination of the amino acid sequenceof the K light chain from a canine IgA (Wasserman et al., Immunochem.,15, 303 (1978)), complete amino-acid sequence of a canine μ chain wasdisclosed (McCumber et al., Mol. Immunol, 16, 565 (1979)), a singlecanine IgG-Aγ chain cDNA and four canine IgG-Aγ chain protein sequenceswere disclosed (Tang et al., Vet. Immunology Immunopathology, 80, 259(2001)). It describes PCR amplification of a canine spleen cDNA librarywith a degenerate oligonucleotide primer designed from the conservedregions of human, mouse, pig, and bovine IgGs. Canine immunoglobulinvariable domains, caninized antibodies, and methods for making and usingthem are disclosed in US Patent Application No. 2004/0181039 and U.S.Pat. Nos. 7,261,890; 6,504,013; 5,852,183; 5,5225,539.

Table 2 below is a list of amino acid sequences of VH and VL regions ofselected caninized anti-NGF antibodies of the disclosure.

TABLE 2 SEQ ID NO: Region 25 72.1 VH 26 72.1 VL 27 73.1 VH 28 73.1 VL 2977.1 VH 30 77.1 VL 31 81.1 VH 32 81.1 VL 33 82.1 VH 34 82.1 VL 35 72.2VH 36 72.2 VL 37 73.2 VH 38 73.2 VL 39 77.2 VH 40 77.2 VL 41 81.2 VH 4281.2 VL 43 82.2 VH 44 82.2 VL 177 81.1B VH 179 72.3 VH 180 72.4 VH 18172.4 VL 182 73.4 VH 183 73.4 VL 184 77.3 VH 185 77.4 VH 186 77.4 VL 18781.4 VH 188 81.4 VL 189 81.2B VH 190 81.4B VH 191 82.3 VL 192 82.4 VH193 82.4 VL 206 81.5B VH 207 81.6B VH

Uses of Anti-NGF Antibodies

Binding proteins as described herein may be used in a method fordetecting the presence of NGF in a sample in vivo or in vitro (e.g., ina biological sample, such as serum, plasma, tissue, biopsy). The invitro method may be used for example to diagnose a disease or disorder,e.g., an NGF-associated disorder. The method includes (i) contacting thesample or a control sample with the anti-NGF antibody or fragmentthereof as described herein; and (ii) detecting formation of a complexbetween the anti-NGF antibody or fragment thereof, and the sample or thecontrol sample, wherein a statistically significant change in theformation of the complex in the sample relative to the control sample isindicative of the presence of the NGF in the sample.

Binding proteins as described herein may be used in a method fordetecting the presence of NGF in vivo (e.g., in vivo imaging in asubject). The method may be used to diagnose a disorder, e.g., anNGF-associated disorder. The method includes: (i) administering theanti-NGF antibody or fragment thereof as described herein to a subjector a control subject under conditions that allow binding of the antibodyor fragment to NGF; and (ii) detecting formation of a complex betweenthe antibody or fragment and NGF, wherein a statistically significantchange in the formation of the complex in the subject relative to thecontrol subject is indicative of the presence of NGF.

Given the ability to bind to NGF, the anti-NGF antibodies, or portionsthereof, or combinations thereof, as described herein may be used asimmunoreagent(s) to detect NGF (e.g., in a biological sample, such asserum or plasma), in a conventional immunoassay, such as an enzymelinked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissueimmunohistochemistry. A method for detecting NGF in a biological sampleinvolves contacting a biological sample with an antibody, or antibodyportion, of the disclosure and detecting either the antibody (orantibody portion) bound to NGF or unbound antibody (or antibodyportion), to thereby detect NGF in the biological sample. The bindingprotein may be directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound antibody.

Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include H ¹⁴C ⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I,¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵Sm.

NGF may alternatively be assayed in biological fluids by a competitionimmunoassay utilizing recombinant NGF standards labeled with adetectable substance and an unlabeled anti-NGF antibody. In this assay,the biological sample, the labeled recombinant NGF standards and theanti-NGF antibody are combined and the amount of labeled rNGF standardbound to the unlabeled antibody is determined. The amount of NGF in thebiological sample is inversely proportional to the amount of labeledrNGF standard bound to the anti-NGF antibody. Similarly, NGF may also beassayed in biological fluids by a competition immunoassay utilizing rNGFstandards labeled with a detectable substance and an unlabeled anti-NGFantibody.

The disclosure thus also contemplates immunoassay reagents, devices andkits including one or more of the presently disclosed binding proteinsfor detecting the presence or amount of NGF in a sample. It iscontemplated for example that an immunoreagent comprising one or more ofthe presently disclosed binding proteins may be provided in the form ofa kit with one or more containers such as vials or bottles, with eachcontainer containing a separate reagent such as an anti-NGF bindingprotein, or a cocktail of anti-NGF binding proteins, detection reagentsand washing reagents employed in the assay. The immunoreagent(s) may beadvantageously provided in a device in which the immunoreagents(s) isimmobilized on a solid support, such as but not limited to a cuvette,tube, microtiter plates or wells, strips, chips or beads. The kit maycomprise at least one container for conducting the assay, and/or abuffer, such as an assay buffer or a wash buffer, either one of whichmay be provided as a concentrated solution, a substrate solution for thedetectable label (e.g., an enzymatic label), or a stop solution.Preferably, the kit comprises all components, i.e., reagents, standards,buffers, diluents, etc., which are necessary to perform the assay. Thekit may contain instructions for determining the presence or amount ofNGF in the sample based on specific binding of the immunoreagent to NGF,in paper form or computer-readable form, such as a disk, CD, DVD, or thelike, and/or may be made available online.

The binding proteins in the kit may be labeled with a detectable labelsuch as those described above including a fluorophore, a radioactivemoiety, an enzyme, a biotin/avidin label, a chromophore, achemiluminescent label, or the like; or the kit may include reagents forcarrying out detectable labeling. The antibodies, calibrators and/orcontrols can be provided in separate containers or pre-dispensed into anappropriate assay format, for example, into microtiter plates.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, enzyme substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine sample). Where appropriate, the kit optionallyalso can contain reaction vessels, mixing vessels, and other componentsthat facilitate the preparation of reagents or the test sample. The kitcan also include one or more instruments for assisting with obtaining atest sample, such as a syringe, pipette, forceps, measured spoon, or thelike. Instructions:

It will be appreciated that the antibodies and antibody portions of thedisclosure are capable of substantially neutralizing NGF activity bothin vitro and in vivo. Accordingly, such antibodies and antibody portionsof the disclosure can also be used to substantially inhibit NGFactivity, e.g., in a cell culture containing NGF, in mammalian subjectshaving NGF with which an antibody of the disclosure cross-reacts. Thedisclosure thus provides a method for inhibiting NGF activity comprisingcontacting NGF with an antibody or antibody portion of the disclosuresuch that NGF activity is substantially inhibited. For example, in acell culture containing, or suspected of containing NGF, an antibody orantibody portion of the disclosure can be added to the culture medium toinhibit NGF activity in the culture.

Accordingly, the disclosure also provides a method for inhibiting NGFactivity comprising contacting NGF with a binding protein such that NGFactivity is substantially inhibited. In another aspect, the disclosureprovides a method for inhibiting NGF activity in a subject sufferingfrom a disorder in which NGF activity is detrimental, comprisingadministering to the subject a binding protein disclosed above such thatNGF activity in the subject is substantially inhibited and treatment isachieved.

The disclosure also provides a method for reducing NGF activity in asubject, such as a subject suffering from a disease or disorder in whichNGF activity is detrimental. The disclosure provides methods forreducing NGF activity in a subject suffering from such a disease ordisorder, which method comprises administering to the subject anantibody or antibody portion of the disclosure such that NGF activity inthe subject is reduced. The subject can be a mammal expressing an NGF towhich an antibody of the disclosure is capable of binding. Still furtherthe subject can be a mammal into which NGF has been introduced (e.g., byadministration of NGF or by expression of an NGF transgene). An antibodyof the disclosure can be administered to a subject in need thereof fortherapeutic purposes.

An antibody of the disclosure can be administered for veterinarypurposes to a non-human mammal expressing an NGF with which the antibodyis capable of binding. For example, an antibody of the disclosure can beadministered for veterinary purposes to a non-human mammal such as adog, horse, cat, or livestock (beef and dairy cattle, swine, sheep,goats, poultry, etc.) expressing an NGF with which the antibody iscapable of binding.

In another aspect, the disclosure provides a method of treating (e.g.,curing, suppressing, ameliorating, delaying or preventing or decreasingthe risk of the onset, recurrence or relapse of) or preventing an NGFassociated disorder, in a subject. The method includes: administering tothe subject a disclosed NGF binding protein (particularly anantagonist), e.g., an anti-NGF antibody or fragment thereof as describedherein, in an amount sufficient to treat or prevent the NGF associateddisorder. The NGF antagonist, e.g., the anti-NGF antibody or fragmentthereof, can be administered to the subject, alone or in combinationwith other therapeutic modalities as described herein.

An antibody of the disclosure can be administered to a non-human mammalexpressing an NGF with which the antibody is capable of binding as ananimal model of human disease. Such animal models may be useful forevaluating the therapeutic efficacy of antibodies of the disclosure(e.g., testing of dosages and time courses of administration).

In another aspect, the antibodies and binding proteins of the disclosureare useful for treating NGF-related diseases and disorders including orinvolving acute or chronic pain. Non-limiting examples of NGF-relateddiseases and disorders include general inflammation, surgical andpostsurgical pain including pain from amputation, dental pain, pain fromtrauma, fracture pain, pain from abscess, neuropathic pain, hyperalgesiaand allodynia, neuropathic pain, post-herpetic neuralgia, diabetesincluding, but not limited to, diabetic neuropathy pain, stroke,thalamic pain syndrome, gout joint pain, osteoarthritis or rheumatoidarthritis pain, rheumatic diseases, lupus, psoriasis, sciatica, painassociated with musculoskeletal diseases including, but not limited to,chronic low back pain, fibromyalgia, sprains, pains associated withsickle cell crises, general headache, migraine, duster headache, tensionheadache, trigeminal neuralgia, dysmenorrhea, endometriosis, ovariancysts, visceral pain, prostatitis, cystitis, interstitial cystitis,erythromelalgia or pain caused by pancreatitis or kidney stones, generalgastrointestinal disorders including, but not limited to, colitis,gastric ulceration and duodenal ulcers, gastroesophageal reflux,dyspepsia, inflammatory bowel disorders, irritable bowel syndrome,inflammatory bladder disorders, incisional pain, pain from burns and/orwounds, ankylosing spondilitis, periarticular pathologies, cancer painincluding, but not limited to, pain from bone metastases and pain fromcancer treatment, and pain from HIV or AIDS. Other examples ofNGF-related diseases and conditions include malignant melanoma,Sjogren's syndrome, rhinitis, bronchial disorders, and asthma, such asuncontrolled asthma with severe airway hyper-responsiveness, intractablecough; and pain from skin diseases or disorders with an inflammatorycomponent such as, but not limited to, sunburn, allergic skin reactions,dermatitis, pruritis, and vitiligo.

The disclosure also provides a method of treating a subject sufferingfrom a disorder in which NGF is detrimental comprising administering abinding protein before, concurrent, or after the administration of asecond agent. In another aspect, the additional therapeutic agent thatcan be co-administered and/or co-formulated with one or more NGFantagonists, (e.g., anti-NGF antibodies or fragments thereof) include,but are not limited to, TNF antagonists; a soluble fragment of a TNFreceptor; ENBREL®; TNF enzyme antagonists; TNF converting enzyme (TACE)inhibitors; muscarinic receptor antagonists; TGF-beta antagonists;interferon gamma; perfenidone; chemotherapeutic agents, methotrexate;leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779; COX2or cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors; TPL-2,MK-2 and NFKB inhibitors; budenoside; epidermal growth factorcorticosteroids; cyclosporine; sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; anti-IL-6 antibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; antibodies or agonists of TNF, CGRP,substance P, bradykinin, MMP-2, MMP-9, MMP-13, LT, IL-1a, IL-11, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24,IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II,GM-CSF, FGF, or PDGF; antibodies of CD2, CD3, CD4, CD8, CD25, CD28,CD30, CD40, CD45, CD69, CD90 or their ligands; FK506; rapamycin;mycophenolate mofetil; ibuprofen; prednisolone; phosphodiesteraseinhibitors; adenosine agonists; antithrombotic agents; complementinhibitors; adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinaseinhibitors; IL-1β converting enzyme inhibitors; TNF converting enzymeinhibitors; T-cell signaling inhibitors; metalloproteinase inhibitors;6-mercaptopurines; angiotensin converting enzyme inhibitors; solublecytokine receptors; soluble p55 TNF receptor; soluble p75 TNF receptor;sIL-IRI; sIL-IRII; sIL-6R; anti-inflammatory cytokines; IL-4; IL-10;IL-11; and TGFβ.

Pharmaceutical Compositions

The antibodies and antibody-portions of the disclosure can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises at least one antibody or antibody portion of the disclosureand a pharmaceutically acceptable carrier. Such compositions can be usedfor example in a method for treating a mammal for a disease or disorderinvolving increased levels of NGF by administering to the mammal aneffective amount of the composition. A pharmaceutical composition mayinclude a therapeutically effective amount of the antibody or antibodyportion. The pharmaceutical compositions as described herein may be usedfor diagnosing, detecting, or monitoring a disorder or one or moresymptoms thereof; preventing, treating, managing, or ameliorating adisorder or one or more symptoms thereof; and/or research. As usedherein, the phrase “increased levels of NGF” refers to a level of NGF ina subject, such as a mammal, that is greater or higher than anestablished or predetermined baseline level of NGF such as, for example,a level previously established for said subject or averaged from a groupof subjects.

A pharmaceutical composition may comprise, for example, a bindingprotein and a pharmaceutically acceptable carrier, excipient or diluent.For example, pharmaceutical compositions may comprise a therapeuticallyeffective amount of one or more of the binding proteins as disclosedherein, together with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant. Thepharmaceutical composition may contain one or more various formulationmaterials for modifying, maintaining or preserving the composition orproperties of the composition, for example, the color, consistency,isotonicity, odor, osmolality, pH, sterility, stability, viscosity andother properties of the composition. Such formulation materials aregenerally well known and many suitable formulation materials aredescribed for example in REMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th)Ed. (A. R. Gennaro, ed.) 1990, Mack Publishing Company. Non-limitingexamples of suitable formulation materials include amino acids (such asglycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. Inaddition, the pharmaceutical composition can also contain one or morepreservatives. Examples of suitable preservatives that can be usedinclude, but are not limited to, methylparaben, propylparaben, benzylalcohol, chlorobutanol, and benzalkonium chloride. Optimalpharmaceutical formulations can be readily determined by one skilled inthe art depending upon, for example, the intended route ofadministration, delivery format and desired dosage.

The pharmaceutical composition may comprise at least one additionaltherapeutic agent for treating a disorder in which NGF activity isdetrimental. The additional agent can be, for example, a therapeuticagent, imaging agent, cytotoxic agent, angiogenesis inhibitors, kinaseinhibitors, co-stimulation molecule blockers, adhesion moleculeblockers, anti-cytokine antibody or functional fragment thereof,methotrexate, cyclosporine, rapamycin, FK506, detectable label orreporter, TNF antagonist, anti-rheumatic, muscle relaxant, narcotic,non-steroid anti-inflammatory drug (NSAID), analgesic, anesthetic,sedative, local anesthetic, neuromuscular blocker, antimicrobial,antipsoriatic, corticosteroid, anabolic steroid, erythropoietin,immunoglobulin, immunosuppressive, growth hormone, hormone replacementdrug, radiopharmaceutical, antidepressant, antipsychotic, stimulant,asthma medication, beta agonist, inhaled steroid, oral steroid,epinephrine or analog, cytokine, or a cytokine antagonist.

The pharmaceutical composition of the present disclosure may have a pHgreater than about 7.0 or between about 7.0 and about 8.0.Alternatively, the pharmaceutical composition may have a pH of betweenabout 7.2 to about 7.8. Still further alternatively, the pH of thepharmaceutical composition may be between about 7.4 to about 7.6. Stillfurther alternatively, the pH of the pharmaceutical composition may beabout 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 7.7, 7.8, 7.9 or 8.0. Withrespect to the pharmaceutical compositions of the present disclosure,there is an increase in degradation, an increase in fragmentation or anincrease in degradation and an increase in fragmentation at a pH of 6.0or less. This finding was surprising as many pharmaceutical compositionscomprising humanized antibodies exhibit an increase in degradation, anincrease fragmentation or an increase in degradation and an increase infragmentation at a pH lower than 5.0 and again at a pH higher than about6.0. Accordingly, most pharmaceutical compositions containing humanizedantibodies are stable at a pH between about 5.0 to about 6.0.

A composition for the release of a binding protein may comprise, forexample, a formulation including an amount of a crystallized bindingprotein, crystallized antibody construct or crystallized antibodyconjugate as disclosed above. The composition may further comprise anadditional ingredient, such as carrier, excipient or diluent, and atleast one polymeric carrier. The polymeric carrier can comprise one ormore polymers selected from the following: poly (acrylic acid),poly(cyanoacrylates), poly (amino acids), poly (anhydrides), poly(depsipeptide), poly (esters), poly (lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly(caprolactone), poly (dioxanone); poly (ethylene glycol), poly((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof. The additionalingredient may be, for example, albumin, sucrose, trehalose, lactitol,gelatin, hydroxypropyl-P-cyclodextrin, methoxypolyethylene glycol andpolyethylene glycol.

The polymeric carrier may be capable of affecting the release of thebinding protein from the composition as described further herein below.Polymeric materials can be used in the formulation of pharmaceuticalcompositions comprising the disclosed binding proteins to achievecontrolled or sustained release of the disclosed binding proteins(Medical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York(1984); Ranger et al., J. Macromol. Sci. Rev. Macromol. Chem., 23:61(1983); Levy et al., Science, 228: 190 (1985); During Qt al, Ann.Neurol., 25: 351 (1989); Howard et al., J. Neurosurg., 7 1: 105 (1989);U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326;PCT Publication Nos. WO 99/15154; and WO 99/20253). Examples of polymersused in sustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. Thepolymer used in a sustained release formulation may be inert, free ofleachable impurities, stable on storage, sterile, and biodegradable. Acontrolled or sustained release system can be placed in proximity of theprophylactic or therapeutic target, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 1 15-138 (1984)).

Controlled release systems are discussed in the review by Langer(Science, 249: 1527-1533 (1990)). Any technique known to one of skill inthe art can be used to produce sustained release formulations comprisingone or more therapeutic agents of the disclosure (U.S. Pat. No.4,526,938; PCT publication Nos. WO 91/05548 and WO 96/20698; Ning etal., Radiotherapy & Oncology, 39: 179-189 (1996), Song et al., PDAJournal of Pharmaceutical Science & Technology, 50: 372-397 (1995);Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater., 24:853-854 (1997); and Lam et al., Proc. Intl. Symp. Control Rel. Bioact.Mater., 24: 759-760 (1997)).

The binding proteins of the present disclosure can be administered by avariety of methods known in the art. For example, the binding proteinsof the present disclosure may be administered by subcutaneous injection,intravenous injection or infusion. Administration can be systemic orlocal. As will be appreciated by the skilled artisan, the route and/ormode of administration may vary depending upon the desired results. Theactive compound may be prepared with a carrier that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are known to thoseskilled in the art. See, e.g., Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,1978.

For example, such pharmaceutical compositions may be administered to asubject by parenteral, intradermal, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal. Methodsof administering a prophylactic or therapeutic agent of the disclosurealso include, but are not limited to, epidural administration,intratumoral administration, and mucosal administration (e.g.,intranasal and oral routes). In addition, pulmonary administration canbe employed, e.g., by use of an inhaler or nebulizer, and formulationwith an aerosolizing agent (U.S. Pat. Nos. 6,019,968; 5,985,320;5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903). The antibodies and antibody portionsdescribed herein can be administered for example using Alkermes AIR®pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).The prophylactic or therapeutic agents may be administered by anyconvenient route, and may be administered together with otherbiologically active agents.

Various delivery systems are known and can be used to administer one ormore disclosed binding proteins or the combination of one or moredisclosed binding proteins and a prophylactic agent or therapeutic agentuseful for preventing, managing, treating, or ameliorating a disorder orone or more symptoms thereof, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe antibody or antibody fragment, receptor-mediated endocytosis (see,e. g., Wu et al., J. Biol. Chem., 262: 4429-4432 (1987)), constructionof a nucleic acid as part of a retroviral or other vector, etc. It maybe desirable to administer the disclosed binding proteins locally to thearea in need of treatment, which may be achieved by, for example, andnot by way of limitation, local infusion, by injection, or by means ofan implant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. An effectiveamount of one or more disclosed binding proteins can be administeredlocally to the affected area to a subject to prevent, treat, manage,and/or ameliorate a disorder or a symptom thereof. Alternatively, aneffective amount of one or more of the disclosed binding proteins isadministered locally to the affected area in combination with aneffective amount of one or more therapies (e. g., one or moreprophylactic or therapeutic agents) other than disclosed bindingproteins of a subject to prevent, treat, manage, and/or ameliorate adisorder or one or more symptoms thereof.

The disclosed binding proteins can be delivered in a controlled releaseor sustained release system such as, for example, an infusion pumpdevice operable to achieve controlled or sustained release of thedisclosed binding proteins (see Langer, supra; Sefton, CRC Crit Ref.Biomed. Eng. 14:20 (1987); Buchwald et al., Surgery, 88: 507 (1980);Saudek et al., N. Engl. J. Med., 321: 574 (1989)).

When a composition as described herein comprises a nucleic acid encodinga binding protein as described herein as a prophylactic or therapeuticagent, the nucleic acid can be administered in vivo to promoteexpression of its encoded prophylactic or therapeutic agent, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., by use of aretroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (Joliot etal., Proc. Natl. Acad. Sci., 88: 1864-1868 (1991)). Alternatively, anucleic acid can be introduced intracellularly and incorporated withinhost cell DNA for expression by homologous recombination.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. For example, a composition may be formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings and companion animals.Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic such aslidocaine to ease pain at the site of the injection.

If the compositions of the disclosure are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art(Remington's Pharmaceutical Sciences and Introduction to PharmaceuticalDosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995)). Fornon-sprayable topical dosage forms, viscous to semi-solid or solid formscomprising a carrier or one or more excipients compatible with topicalapplication and having a dynamic viscosity greater than water aretypically employed. Suitable formulations include, without limitation,solutions, suspensions, emulsions, creams, ointments, powders,liniments, salves, and the like, which are, if desired, sterilized ormixed with auxiliary agents (e.g., preservatives, stabilizers, wettingagents, buffers, or salts) for influencing various properties, such as,for example, osmotic pressure. Other suitable topical dosage formsinclude sprayable aerosol preparations wherein the active ingredient, incombination with a solid or liquid inert carrier, is packaged in amixture with a pressurized volatile (e.g., a gaseous propellant, such asFreon) or in a squeeze bottle. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well-known in the art.

The pharmaceutical composition of the present disclosure can have ahalf-life of from about 8 days to about 15 days when dosed intravenouslyor subcutaneously. Alternatively, the pharmaceutical composition of thepresent invention can have a half-life of from about 10 days to about 13days. Still further alternatively, the pharmaceutical composition of thepresent invention can have a half-life of about 8 days, such as about8.5 days, about 9 days, such as about 9.5 days, about 10 days, such asabout 10.5 days, about 11 days, such as about 11.5 days, about 12 days,about 12.5 days, about 13 days, such as about 13.5 days, about 14 days,such as about 14.5 days, or about 15 days.

If the method of the disclosure comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present disclosure can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the disclosure comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gelcaps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excepients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the disclosure may comprise pulmonary administration,e.g., by use of an inhaler or nebulizer, of a composition formulatedwith an aerosolizing agent (U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903). For example, an antibody of the disclosure,combination therapy, and/or composition of the disclosure may beadministered using Alkermes AIR® pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass.).

The method of the disclosure may comprise administration of acomposition formulated for parenteral administration by injection (e.g.,by bolus injection or continuous infusion). Formulations for injectionmay be presented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the disclosure may additionally comprise administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the disclosure encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions, such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

The ingredients of the disclosed compositions may be supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or a substantially water-free concentrate in ahermetically sealed container such as an ampoule or sachette which mayindicate the quantity of active agent. Where the mode of administrationis infusion, the disclosed compositions can be dispensed with aninfusion solution containing sterile pharmaceutical grade solution suchas water or saline. Where the mode of administration is by injection, anampoule of sterile solution such as water or saline can be provided sothat the ingredients may be mixed prior to administration.

In particular, the disclosure also provides that one or more ofdisclosed binding proteins or pharmaceutical compositions thereof ispackaged in a hermetically sealed container such as an ampoule orsachette which may indicate the quantity of the agent. One or more ofthe disclosed binding proteins or pharmaceutical compositions thereofmay be supplied as a dry sterilized lyophilized powder or substantiallywater-free concentrate in a hermetically sealed container and can bereconstituted (e.g., with water or saline) to the appropriateconcentration for administration to a subject. One or more of thedisclosed binding proteins or pharmaceutical compositions thereof may besupplied as a dry sterile lyophilized powder in a hermetically sealedcontainer at a unit dosage of at least about 0.5 mg, 1 mg, 2 mg, 4 mg, 5mg, 10 mg, 15 10 mg, 25 mg, 35 mg, 45 mg, 50 mg, 75 mg, or 100 mg. Thelyophilized disclosed binding proteins or pharmaceutical compositionsthereof may be stored at any suitable temperature, such as, for example,between about 2° C. and about 8° C. and may be stored in its originalcontainer. The disclosed binding proteins or pharmaceutical compositionsthereof may be administered within about 1 week, within about 5 days,within about 72 hours, within about 48 hours, within about 24 15 hours,within about 12 hours, within about 6 hours, within about 5 hours,within about 3 hours, or within about 1 hour after being reconstituted.Alternatively, one or more of the disclosed binding proteins orpharmaceutical compositions thereof may be supplied in liquid form in ahermetically sealed container which may indicate the quantity andconcentration of the agent. The liquid form of the administeredcomposition may be supplied in a hermetically sealed container atconcentrations of at least about 0.01 mg/mL, at least about 0.05 mg/mL,at least about 0.1 mg/mL, at least about 0.2 mg/mL, at least about 0.25mg/ml, at least about 0.5 mg/ml, at least about 1 mg/ml, at least about2.5 mg/ml, at least about 5 mg/ml, at least about 8 mg/ml, at leastabout 10 mg/ml, at least about 15 mg/kg, at least about 25 mg/ml, atleast about 50 mg/ml, at least about 75 mg/ml, or at least about 100mg/ml. The liquid form may be stored at any suitable temperature such asbetween about 2° C. and about 8° C. and may be stored in its originalcontainer.

The binding proteins of the disclosure can be incorporated into apharmaceutical composition suitable for parenteral administration. Inone aspect, binding proteins are prepared as an injectable solutioncontaining between about 0.1 and about 250 mg/ml antibody. Theinjectable solution can be composed of either a liquid or lyophilizeddosage form in a flint or amber vial, ampoule or pre-filled syringe. Thebuffer can be any suitable buffer such as L-histidine or a phosphatebuffer saline at a concentration of about 1-50 mM, or about 5-10 mM.Other suitable buffers include, but are not limited to, sodiumsuccinate, sodium citrate, sodium phosphate and potassium phosphate.Buffers may be used to modify the toxicity of the pharmaceuticalcomposition. For example, sodium chloride can be used to modify thetoxicity of the binding protein solution at a concentration of fromabout 0.1 and about 300 mM, such as about 150 mM saline to modify thetoxicity of a liquid dosage form.

Cryoprotectants, such as sucrose, can be included in a lyophilizeddosage form at a concentration of about 0.1 to about 10% or from about0.5 to about 1.0% may be used. Other suitable cryoprotectants include,but are not limited to, trehalose and lactose. Bulking agents, such asmannitol, can be included in a lyophilized dosage form at aconcentration of about 1 to about 10%, or from about 2 to about 4%.Stabilizers, such as L-Methionine, can be used in both liquid andlyophilized dosage forms at a concentration of about 1 to about 50 mM,or about 5 to about 10 mM). Other suitable bulking agents include, butare not limited to, glycine and arginine. Surfactants, such aspolysorbate-80, can be included in both liquid and lyophilized dosageforms at a concentration of about 0.001 to about 0.05% or about 0.005 toabout 0.01%. Additional surfactants include, but are not limited to,polysorbate 20 and BRIJ surfactants.

An exemplary pharmaceutical formulation or composition of the presentdisclosure may be a liquid pharmaceutical composition having a pHbetween about 7.4 to about 8.0. The liquid pharmaceutical compositioncomprises about 5 mg/ml to about 50 mg/ml of an antibody comprising aheavy chain variable region comprising an amino acid sequence having asequence of SEQ ID NO: 37 and a light chain variable region comprisingan amino acid sequence comprising a sequence of SEQ ID NO: 38. Theliquid pharmaceutical composition further comprises at least one buffer(such as, phosphate buffer saline, tris or histidine). The molarity ofbuffer that can be used can be from about 1 mM to about 60 mM.Optionally, said pharmaceutical composition or formulation can alsocontain at least one preservative, such as, methylparaben,propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride.The amount of preservative that can be used can be from about 0.01percent by volume to about 5.0% by volume depending on the preservativeused.

Another exemplary pharmaceutical formulation or composition of thepresent disclosure may be a liquid pharmaceutical composition comprisinga pH between about 7.4 to about 8.0. The liquid pharmaceuticalcomposition comprises about 5 mg/ml to about 50 mg/ml of an antibodycomprising a heavy chain variable region comprising an amino acidsequence having a sequence of SEQ ID NO: 192 and a light chain variableregion comprising an amino acid sequence comprising a sequence of SEQ IDNO: 193. The liquid pharmaceutical composition further comprises atleast one buffer (such as, phosphate buffer saline, tris or histidine).The molarity of buffer that can be used can be from about 1 mM to about60 mM. Optionally, said pharmaceutical composition or formulation canalso contain at least one preservative, such as, methylparaben,propylparaben, benzyl alcohol, chlorobutanol or benzalkonium chloride.The amount of preservative that can be used can be from about 0.01percent by volume to about 5.0% by volume depending on the preservativeused.

The compositions of this disclosure may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The form of the disclosed composition may depend on the intended mode ofadministration and therapeutic application. The disclosed compositionsmay be in the form of injectable or infusible solutions, such ascompositions similar to those used for passive immunization of humanswith other antibodies. The mode of administration may be parenteral(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Thedisclosed binding proteins may be administered by intravenous infusionor injection, or by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other suitableordered structure such as those suitable to high drug concentration.Sterile injectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Dispersions may be prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the methods of preparation include, but are not limited to, vacuumdrying and spray-drying that yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption such as, for example, monostearate saltsand gelatin.

An antibody or antibody portion of the disclosure may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the disclosure by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

The disclosed binding proteins may be co-administered with other activecompounds which may also be incorporated into the disclosedcompositions. An antibody or antibody portion of the disclosure may beco-formulated with and/or co-administered with one or more additionaltherapeutic agents that are useful for treating disorders in which NGFactivity is detrimental. For example, an anti-NGF antibody or antibodyportion of the disclosure may be co-formulated and/or co-administeredwith one or more additional antibodies that bind other targets (e.g.,antibodies that bind other cytokines or that bind cell surfacemolecules). Furthermore, one or more disclosed binding proteins may beused in combination with two or more of the foregoing therapeuticagents. Such combination therapies may, for example, enable the use oflower dosages of the administered therapeutic agents, thus avoidingpossible toxicities or complications associated with the variousmonotherapies.

An antibody to NGF or fragment thereof may be formulated with a vehiclethat extends the half-life of the binding protein. Suitable vehiclesknown in the art include, but are not limited to, the Fc domain,polyethylene glycol, and dextran. Such vehicles are described, e.g., inU.S. application Ser. No. 09/428,082 and published PCT Application No.WO 99/25044.

Isolated nucleic acid sequences comprising nucleotide sequences encodingdisclosed binding proteins or another prophylactic or therapeutic agentof the disclosure may be administered to treat, prevent, manage, orameliorate a disorder or one or more symptoms thereof by way of genetherapy. Gene therapy refers to therapy performed by the administrationto a subject of an expressed or expressible nucleic acid, wherein thenucleic acids produce their encoded antibody or prophylactic ortherapeutic agent of the disclosure that mediates a prophylactic ortherapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present disclosure. For general reviews of the methodsof gene therapy, see Goldspiel et al., Clinical Pharmacy, 12: 488-505(1993); Wu et al., Biotherapy, 3: 87-95 (1991); Tolstoshev, Ann. Rev.Pharmacol. Toxicol., 32: 573-596 (1993); Mulligan, Science, 260: 926-932(1993); and Morgan et al., Ann. Rev. Biochem., 62: 191-217 (1993);TIBTECH, 11(5): 155-215 (1993). Methods commonly known in the art ofrecombinant DNA technology which can be used are described in, forexample, Ausubel et al. (eds.), Current Protocols in Molecular Biology,John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY (1990). Detaileddescriptions of various methods of gene therapy are disclosed inUS20050042664A1.

Antibodies of the disclosure, or antigen binding portions thereof, canbe used alone or in combination to treat NGF related diseases. It shouldbe understood that the antibodies of the disclosure or antigen bindingportion thereof can be used alone or in combination with an additionalagent, e.g., a therapeutic agent, said additional agent being selectedby the skilled artisan for its intended purpose. For example, theadditional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present disclosure. The additional agent also can be an agentthat imparts a beneficial attribute to the therapeutic composition e.g.,an agent which affects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this disclosure are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this disclosure, can be the antibodies of the present disclosureand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

Combinations include non-steroidal anti-inflammatory drug(s) alsoreferred to as NSAIDS which include drugs like ibuprofen. Othercombinations are corticosteroids including prednisolone; the well-knownside-effects of steroid use can be reduced or even eliminated bytapering the steroid dose required when treating patients in combinationwith the anti-NGF antibodies of this disclosure. Non-limiting examplesof therapeutic agents for rheumatoid arthritis or pain with which anantibody, or antibody portion, of the disclosure can be combined includethe following: cytokine suppressive anti-inflammatory drug(s) (CSAIDs);antibodies to or antagonists of cytokines or growth factors, forexample, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15,IL-16, IL-18, IL-21, interferons, EMAP-II, GM-CSF, FGF, and PDGF.Antibodies of the disclosure, or antigen binding portions thereof, canbe combined with antibodies to cell surface molecules such as CD2, CD3,CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2),CD90, CTLA or their ligands including CD154 (gp39 or CD40L).

Combinations of therapeutic agents may interfere at different points inthe autoimmune and subsequent inflammatory cascade; examples include TNFantagonists like chimeric, humanized or human TNF antibodies, D2E7, (PCTPublication No. WO 97/29131), CA2 (Remicade™), CDP 571, and soluble p55or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG (Enbrel™) orp55TNFR1gG (Lenercept), and also TNFa converting enzyme (TACE)inhibitors; similarly other IL-1 inhibitors (Interleukin-1-convertingenzyme inhibitors, IL-IRA etc.) may be effective for the same reason.Other combinations include Interleukin 11.

The antibodies of the disclosure, or antigen binding portions thereof,may also be combined with agents, such as methotrexate, 6-MP,azathioprine sulphasalazine, mesalazine, olsalazinechloroquinine/hydroxychloroquine, pencillamine, aurothiomalate(intramuscular and oral), azathioprine, cochicine, corticosteroids(oral, inhaled and local injection), beta-2 adrenoreceptor agonists(salbutamol, terbutaline, salmeteral), xanthines (theophylline,aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium andoxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signaling by proinflammatory cytokines such as TNFor IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1βconverting enzyme inhibitors, TNFa converting enzyme (TACE) inhibitors,T-cell signaling inhibitors such as kinase inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors and derivativesthereof (e.g. soluble p55 or p75 TNF receptors and the derivativesp75TNFRIgG (Enbrel™ and p55TNFRIgG (Lenercept)), sIL-IRI, sIL-IRII,sIL-6R), anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 andTGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib,etanercept, infliximab, naproxen, valdecoxib, sulfasalazine,methylprednisolone, meloxicam, methylprednisolone acetate, gold sodiumthiomalate, aspirin, triamcinolone acetonide, propoxyphenenapsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac,diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodonebitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra,human recombinant, tramadol hcl, salsalate, sulindac,cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium,prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin,glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodonehcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium,omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,Roflumilast, IC-485, CDC-801, and Mesopram. Other combinations includemethotrexate or leflunomide and in moderate or severe rheumatoidarthritis cases, cyclosporine. The antibodies of the disclosure, orantigen binding portions thereof, may also be combined with agents, suchas cancer chemotherapeutics, antimicrobials, anti-inflammatories, andanthelmintics used in animals.

The NSAID may be any non-steroidal anti-inflammatory compound. NSAIDsare categorized by virtue of their ability to inhibit cyclooxygenase.Cyclooxygenase 1 and cyclooxygenase 2 are two major isoforms ofcyclooxygenase and most standard NSAIDs are mixed inhibitors of the twoisoforms. Most standard NSAIDs fall within one of the following fivestructural categories: (1) propionic acid derivatives, such asibuprofen, naproxen, naprosyn, diclofenac, and ketoprofen; (2) aceticacid derivatives, such as tolmetin and slindac; (3) fenamic acidderivatives, such as mefenamic acid and meclofenamic acid; (4)biphenylcarboxylic acid derivatives, such as diflunisal and flufenisal;and (5) oxicams, such as piroxim, sudoxicam, and isoxicam. Another classof NSAID has been described which selectively inhibit cyclooxygenase 2.Cox-2 inhibitors have been described (U.S. Pat. Nos. 5,616,601;5,604,260; 5,593,994; 5,550,142; 5,536,752; 5,521,213; 5,475,995;5,639,780; 5,604,253; 5,552,422; 5,510,368; 5,436,265; 5,409,944; and5,130,311). Certain exemplary COX-2 inhibitors include celecoxib(SC-58635), rofecoxib, DUP-697, flosulide (CGP-28238), meloxicam,6-methoxy-2 naphthylacetic acid (6-MNA), MK-966, nabumetone (prodrug for6-MNA), nimesulide, NS-398, SC-5766, SC-58215, T-614; or combinationsthereof.

The NGF antagonist and/or an additional therapeutic agent, such asNSAID, can be administered to a subject via any suitable route. Forexample, they can be administered together or separately, and/orsimultaneously and/or sequentially, orally, intravenously, sublingually,subcutaneously, intraarterially, intramuscularly, rectally,intraspinally, intrathoracically, intraperitoneally, intraventriculariy,sublingually, transdermally or by inhalation. Administration can besystemic, e.g., intravenous, or localized. The nerve growth factorantagonist and the additional therapeutic agent may be present togetherwith one or more pharmaceutically acceptable carriers or excipients, orthey may be present in separate compositions. In another aspect, theinvention provides a synergistic composition of an NGF antagonist and anNSAID.

The pharmaceutical compositions of the disclosure may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the disclosure. Dosageregimens may be adjusted to provide the optimum desired response (e.g.,a therapeutic or prophylactic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe disclosure is about 0.001 to about 20 mg/kg or about 0.001 to about10 mg/kg. It is to be noted that dosage values may vary with the typeand severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the disclosuredescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the disclosure or the embodimentsdisclosed herein. Having now described the present disclosure in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the disclosure.

EXAMPLES

The following examples are provided for exemplary guidance to make anduse the disclosed binding proteins and pharmaceutical compositionsthereof according to the inventive subject matter. However, it should berecognized that numerous modifications may be made without departingfrom the inventive concept presented herein.

Example 1 Immunization of Mice with NGF

To generate mouse anti-NGF mAbs, female A/J mice were immunizedsubcutaneously with 25 μg of human β NGF (R&D Systems catalog#256-GF/CF) in CFA. Animals were boosted every three weeks with 25 μghuman β NGF in IFA. Four days prior to fusion, the mice were boostedwith 10 μg of human β NGF in sterile saline intravenously. Spleen cellsfrom the immunized mouse were fused with SP2/0-Agl4 myeloma cells at a5:1 ratio spleen to SP2/0 cells, using standard techniques. Seven to tendays post fusion, when macroscopic colonies were observed; supernatantswere tested in a capture ELISA format for binding to biotinylated humanor rat β NGF. ELISA-positive wells were expanded to 24 well plates andtested for binding to biotinylated rat β NGF. Supernatants fromhybridoma cell lines testing positive for both human and rat NGF wereevaluated in a bioassay format. Cell lines of interest were cloned bylimiting dilution to isolate an NGF-specific mouse monoclonal antibody.

Example 2 Screening Hybridoma Supernatants to Identify Secreted Anti-NGFMAbs A. Indirect Binding ELISA

To determine if anti-NGF mAbs were present in hybridoma supernatants,ELISA plates were coated with goat anti-murine IgG Fc (JacksonImmunoResearch, cat #115-005-164) and incubated overnight at 4° C. Theplates were washed three times with wash buffer. The plates were blockedwith 200 μl of 2% milk and incubated for 1 hour at room temperature. Theplates were washed as above. Hybridoma supernatants were diluted 5-fold,25-fold, 125-fold and 1625-fold with PBS and then added to the platewells and incubated for 1 hour at room temperature. The positive controlwas crude sera (diluted 1:500 with PBS) isolated from a β NGF immunizedmouse and the negative control was hybridoma supernatant derived from amouse immunized with an antigen other than NGF. The plates were washedand then 50 μl of biotinylated human or rat β NGF at 50 ng/ml was addedand incubated for 1 hour at room temperature. The plates were washed.Streptavidin-HRP (Thermo, cat #21 126) conjugate was diluted at 10,000and added to the plates. The plates were incubated for 30 minutes atroom temperature. The plates were washed and then TMB substrate(Invitrogen, catalog #00-2023) was added. The reaction was stopped using2N H₂S0₄ (VWR, catalog #BDH3500-1). The absorbance at 450 nm was read ona Spectromax 2E plate reader (Molecular Devices); these absorbancereadings are shown in Tables 1 and 2. The numerical value indicatesbinding of mouse anti-NGF antibodies to biotinylated human or rat β NGF.This data indicates that several hybridoma supernatants containedanti-NGF antibodies.

TABLE 3 Biotinylated Human NGF Indirect Binding ELISA data Supernatantdilution (fold) 30F11 23F1 22E1 3C3 16B9 17G6 23H2 25E5 29E6 7H1 19C130A1  5 1.066 1.143 1.288 1.137 0.821 1.122 0.913 1.299 1.196 1.1550.936 1.09  25 1.005 1.171 1.255 1.108 0.644 1.127 0.529 1.254 1.1271.159 0.555 0.926 125 0.873 0.979 0.772 0.948 0.34 1.017 0.191 0.9880.889 1.002 0.234 0.507 625 0.436 0.696 0.296 0.571 0.107 0.713 0.0850.512 0.426 0.673 0.1  0.223 Supernatant dilution (fold) 29A7 27A5 26D526H12 23D7 22A9 22G3 21D4 3E9 3F9 2G11 1D6  5 1.198 1.116 0.954 0.9431.087 0.707 0.662 1.154 1.167 0.974 1.038 0.545  25 1.092 0.887 0.9030.794 1.06  0.549 0.498 1.042 0.996 0.694 0.992 0.457 125 0.762 0.3950.823 0.381 0.857 0.348 0.24  0.899 0.655 0.323 0.819 0.164 625 0.2930.174 0.542 0.135 0.489 0.168 0.126 0.543 0.298 0.145 0.486 0.066Supernatant dilution (fold) 4B6 8E4 9E2 9H2 20B10 14G6 12H12 11D1  51.252 1.294 1.126 1.167 1.098 1.274 1.222 0.642  25 1.131 1.076 1.0850.915 0.997 1.206 1.083 0.497 125 0.768 0.595 0.938 0.395 0.576 0.9560.741 0.275 625 0.341 0.25  0.605 0.171 0.143 0.598 0.363 0.117Supernatant dilution Positive (fold) control 4E2 12D6 1D10 2D8 3F7 4F114H2 5D8 5G9 6B2 6F10  3 1.018 1.078 0.985 1.105 1.046 1.282 1.192 1.0130.79 1.052 1.231 1.096 15 0.981 0.991 0.844 0.963 0.868 1.166 1.016 0.80.654 0.919 0.939 1.045 75 1.02  0.705 0.501 0.655 0.436 1.049 0.7020.447 0.42 0.534 0.505 0.999 Supernatant dilution Negative Negative(fold) 6H2 7C10 7G1 8G9 10A12 10B6 11A9 12A5 12F6 13E3 14A9 control  31.322 0.745 0.233 0.849 0.192 1.135 0.056 0.725 1.003 1.003 1.107 0.05415 1.221 0.378 0.106 0.548 0.089 1.088 0.051 0.401 0.944 0.881 1.0820.053 75 0.791 0.151 0.066 0.22  0.06  0.872 0.05 0.183 0.681 0.4630.951 0.051

TABLE 4 Biotinylated Rat NGF Indirect Binding ELISA data Supernatantdilution (fold) 30F11 23F1 22E1 3C3 16B9 17G6 23H2 25E5 29E6 7H1 19C130A1  5 0.694 0.764 1.054 0.698 0.443 0.749 0.670 1.091 0.677 0.7330.660 0.690  25 0.734 0.767 0.936 0.729 0.350 0.758 0.412 1.099 0.6550.664 0.462 0.681 125 0.603 0.737 0.557 0.628 0.218 0.751 0.176 0.8030.523 0.603 0.197 0.445 625 0.361 0.528 0.229 0.520 0.094 0.567 0.0830.396 0.261 0.401 0.088 0.180 Supernatant dilution (fold) 29A7 27A5 26D526H12 23D7 22A9 22G3 21D4 3E9 3F9 2G11 1D6  5 0.967 0.610 0.611 0.5380.684 0.508 0.521 0.787 1.098 0.633 0.705 0.327  25 0.907 0.514 0.5710.368 0.775 0.417 0.384 0.760 0.945 0.502 0.669 0.278 125 0.441 0.2360.516 0.169 0.654 0.240 0.209 0.671 0.530 0.264 0.588 0.132 625 0.2240.113 0.413 0.082 0.396 0.117 0.107 0.453 0.219 0.117 0.353 0.063Supernatant dilution (fold) 4B6 8E4 9E2 9H2 20B10 14G6 12H12 11D1  50.607 0.685 0.632 0.453 0.472 0.755 0.676 0.122  25 0.508 0.518 0.5590.310 0.431 0.739 0.571 0.095 125 0.438 0.317 0.529 0.157 0.261 0.6650.357 0.076 625 0.234 0.150 0.382 0.085 0.108 0.424 0.173 0.060Supernatant dilution Positive (fold) control 4E2 12D6 1D10 2D8 3F7 4F114H2 5D8 5G9 6B2 6F10  3 0.773 0.777 0.459 1.023 0.590 1.097 0.952 0.9450.565 0.952 1.122 0.937 15 0.736 0.651 0.379 0.877 0.599 1.125 0.6900.684 0.467 0.767 0.876 1.005 75 0.760 0.471 0.210 0.548 0.323 1.0440.576 0.348 0.294 0.406 0.453 0.849 Supernatant dilution Negative (fold)6H2 7C10 7G1 8G9 10A12 10B6 11A9 12A5 12F6 13E3 14A9 control  3 1.1080.541 0.197 0.681 0.145 0.440 0.058 0.521 0.904 0.786 0.845 0.055 150.860 0.275 0.093 0.396 0.077 0.784 0.052 0.334 0.810 0.737 0.777 0.05375 0.603 0.115 0.061 0.155 0.060 0.727 0.051 0.153 0.565 0.413 0.5820.052

B. TrkA Binding ELISA

To determine if anti-NGF mAbs in hybridoma supernatants blocked NGF frombinding to the TrkA receptor, ELISA plates were coated with goatanti-human IgG Fc (Jackson ImmunoResearch, cat #109-005-008) at 2 μg/mlin PBS and incubated over night at 4° C. The plates were washed threetimes with PBS/Tween. The plates were blocked with 200 μl/well of 2%milk in PBS for 1 hour at room temperature. The plates were washed threetimes as above. Rat TrkA/Fc chimera (R&D Systems, catalog #1056-TK) wasadded at 1 μg/ml (50 μl/well) in PBS/0.1% BSA and then incubated for 1hour at room temperature. Biotinylated human NGF was titered andpre-incubated with anti-NGF antibody supernatants diluted 1-fold,5-fold, and 25-fold, or purified anti-NGF mAbs diluted to 0.08, 0.4, 2,or 10 μg/ml for 1 hour at room temperature on a plate shaker. Thenegative control was unrelated conditioned supernatant. The positivecontrol was sera from a mouse immunized with NGF. The plates were washedand then 50 μl of each biotinylated NGF/Ab mix was added to theappropriate wells. The plates were incubated for 1 hour at roomtemperature. The plates were washed. 50 μl of streptavidin-HRP (Thermo,cat #21 126) was added at 10,000 dilution. The plates were incubated for30 min at room temperature. The plates were washed. 50 μl of TMB(Invitrogen, cat #00-2023) was added and the reaction was stopped using2N H₂S0₄ (VWR, cat #BDH3500-1). The absorbance at 450 nm was read on aSpectromax 2E plate reader (Molecular Devices), and the absorbancereadings are shown in Table 5. The numerical value indicates binding ofbiotinylated human β NGF to rat TrkA/Fc chimera. This data indicatesthat several hybridoma supernatants contained anti-NGF receptor-blockingantibodies.

TABLE 5 Rat TrkA Inhibition Binding ELISA Data for Anti-NGF HybridomaSupernatants Supernatant dilution Negative Positive (fold) controlcontrol 30F11 23F1 22E1 3C3 16B9 17G6 23H2 25E5 29E6 7H1 1 0.465 0.0500.158 0.108 0.357 0.146 0.142 0.091 0.379 0.304 0.291 0.217 5 0.4560.055 0.210 0.140 0.429 0.195 0.249 0.123 0.622 0.354 0.600 0.419 25 0.462 0.102 0.331 0.276 0.558 0.345 0.409 0.210 0.418 0.505 0.881 0.758Supernatant dilution (fold) 19C1 30A1 29A7 27A5 26D5 26H12 23D7 22A922G3 21D4 3E9 3F9 1 0.285 0.148 0.427 0.444 0.063 0.344 0.131 0.3220.150 0.133 0.328 0.186 5 0.567 0.281 0.462 0.800 0.076 0.621 0.2120.362 0.211 0.242 0.416 0.295 25  0.686 0.464 0.502 0.680 0.101 0.6650.393 0.453 0.337 0.404 0.682 0.498 Supernatant Dilution (fold) Neg Pos2G11 1D6 4B6 8E4 9E2 9H2 1 0.372 0.052 0.138 0.226 0.169 0.273 0.1030.380 5 0.336 0.073 0.205 0.281 0.287 0.669 0.125 0.604 25  0.318 0.2280.328 0.343 0.424 0.693 0.151 0.521 Supernatant dilution (fold) 20B1014G6 12H12 11D1 19A12 2B12 PBS PBS 1 0.166 0.113 0.060 0.101 0.100 0.0650.295 0.315 5 0.200 0.192 0.099 0.152 0.170 0.070 0.334 0.297 25  0.2890.334 0.190 0.264 0.295 0.095 0.306 0.289 Supernatant dilution −ve +ve+ve +ve (fold) contrl contrl contrl contrl 13E3 14A9 4E2 12D6 1D10 2D83F7 4F11 1 0.386 0.112 0.145 0.104 0.400 0.121 0.283 0.145 0.248 0.3590.056 0.286 5 0.388 0.164 0.234 0.140 0.383 0.208 0.290 0.211 0.3120.588 0.083 0.356 25  0.386 0.308 0.488 0.216 0.497 0.376 0.334 0.3640.447 0.497 0.149 0.541 Supernatant dilution (fold) 4H2 5D8 5G9 6B2 6F106H2 7C10 8G9 10B6 12A5 12F6 PBS 1 0.396 0.363 0.344 0.096 0.206 0.4000.230 0.409 0.329 0.306 0.172 0.436 5 0.457 0.398 0.387 0.215 0.2120.523 0.489 0.473 0.364 0.328 0.227 0.351 25  0.606 0.504 0.473 0.4510.242 0.738 0.487 0.413 0.399 0.406 0.324 0.338C. SureFire Cellular Phospho-ERK (pERK) Assay

To determine if anti-NGF mAbs in hybridoma supernatants blockeddownstream signaling as a result of blocking NGF from binding to TrkA,Neuroscreen-1 cells (Thermo Fisher Scientific) were grown on collagenI-coated flasks in RPMI medium supplemented with 10% horse serum, 5%FBS, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, and 10 mMHEPES at 37° C. in a humidified atmosphere at 95% air and 5% CO₂. Forthe ERK phosphorylation assay, 5×10⁴ cells were seeded in each well of a96-well plate coated with collagen I (Becton Dickinson). Cells were thenserum starved for 24 hours before stimulation. 130 pM human β NGF (R&DSystems catalog #256-GF/CF) was mixed into diluted hybridomasupernatants (to achieve a final supernatant dilution (fold) of 10-fold,100-fold, 500-fold or 1,000-fold) and mixtures were pre-incubated for 15min at 37° C. before being added to the cells. Each diluted hybridomasupernatant was tested in quadruplicate. After 5 min of stimulation, themedium was removed and replaced with SureFire™ AlphaScreen cell lysis(PerkinElmer). Cell lysates were then processed according to themanufacturer's instructions and fluorescence signals quantified using anEn Vision plate reader (PerkinElmer); the fluorescence data issummarized in Table 6. The numerical value indicates ERK phosphorylationdue to TrkA signaling in the presence of human β NGF and is expressed asthe percentage of signal vs. maximum signal. The maximum signal isdefined as 100% response from cells showing ERK phosphorylation in thepresence of only β NGF (no hybridoma supernatant). This data indicatesthat several hybridoma supernatants contained neutralizing anti-NGFantibodies.

TABLE 6 SureFire pERK Assay Data Generated with Anti-NGF mAb HybridomaSupernatants Supernatant dilution (fold) 23F1 17G6 30F11 3C3 100 5 2 4 22 0 1 1 5 2 2 0 5 2 5 3 1000 5 3 4 3 2 2 2 1 4 2 4 3 6 3 5 4 5000 8 7 88 13 7 15 8 30 26 28 25 24 25 22 23 10000 35 33 32 32 44 25 43 23 65 4556 42 57 52 68 62 Supernatant dilution (fold) 2B12 21D4 4B6 22G3 100 0 0−1 0 4 3 5 2 5 4 1 0 13 7 7 2 1000 0 0 0 0 5 3 5 3 1 0 2 1 3 2 4 2 500018 16 21 17 11 7 12 8 8 8 7 8 23 18 25 20 10000 51 43 49 41 38 23 37 2330 34 30 35 51 45 47 43 Supernatant dilution (fold) 2G11 14G6 16B9 19A12100 5 2 6 3 4 3 0 −1 3 3 3 2 2 1 2 1 1000 6 3 6 3 −1 0 0 0 65 57 70 60 32 3 2 5000 14 8 14 8 7 7 8 7 72 63 73 62 47 36 46 32 10000 44 30 74 4838 36 36 36 76 62 77 62 69 55 81 65 Supernatant dilution (fold) 30A126D5 23D7 23H2 100 0 0 1 0 0 0 0 0 2 2 −1 0 47 41 44 44 1000 1 2 2 2 1 11 1 1 0 1 1 86 80 79 82 5000 40 41 42 40 37 30 35 30 59 48 56 54 85 8085 83 10000 62 67 63 64 64 52 80 71 74 63 70 60 85 84 82 84 Supernatantdilution (fold) 9E2 20B10 12H12 11D1 100 3 3 3 4 2 1 1 1 3 3 3 2 69 7570 78 1000 5 7 5 8 1 1 1 0 30 30 30 32 71 84 72 85 5000 37 57 36 55 2028 19 29 62 64 60 61 76 78 80 76 10000 55 69 56 67 55 71 73 84 69 78 7677 89 95 102 101

D. PathHunter Assay

To determine if anti-NGF mAbs in hybridoma supernatants blockeddownstream signaling as a result of blocking NGF from binding to TrkA,the PathHunter U20S stable cell line stably expressing the NGF receptorTrkA and the co-activator protein SHC1 fused to complementing fragmentsof β-galactosidase was purchased from DiscoveRx. Cells were grown in MEMmedia supplemented with 10% FBS, 100 units/ml penicillin/streptomycin, 2mM L-glutamine, 500 μg/ml Geneticin G418, and 250 μg/ml Hygromycin at37° C. in a humidified atmosphere at 95% air and 5% CO₂. Sixteen hoursbefore the assay, 2×10⁴ cells were seeded in each well of a 96-wellhalf-volume black plate in 40 μl of MEM media supplemented with 0.5%horse serum. 440 pM human β NGF (R and D Systems catalog #256-GF/CF) wasmixed into diluted hybridoma supernatants (to achieve a finalsupernatant dilution of 10-fold, 100-fold, 500-fold or 1,000-fold) andmixtures were pre-incubated for 15 min at 37° C. before being added tothe cells. Cell plates were incubated for 5 min at room temperaturebefore stimulation with 10 μl per well of NGF/antibody mixture. After 3hours of cell induction at room temperature, 25 μl of PathHunterdetection reagent was added to each well according to the manufacturersinstructions. The chemiluminescent signal was detected 1 hour laterusing a TopCount plate reader (PerkinElmer); the chemiluminescencesignal data is shown in Table 7. The numerical value indicatesβ-galactosidase generation due to TrkA signaling in the presence ofhuman β NGF and is expressed as the percentage of signal vs. maximumsignal. The maximum signal is defined as 100% response from cellsshowing in the presence of β-galactosidase generation in the presence ofonly β NGF (no hybridoma supernatant). This data indicates that severalhybridoma supernatants contained neutralizing anti-NGF antibodies.

TABLE 7 PathHunter Data Generated with Hybridoma SupernatantsSupernatant dilution (fold) 30F11 23F1 3C3 16B9 17G6 19A12 100 21 22 5 516 29 19 24 10 14 25 28 1000 42 37 23 13 40 46 117 114 23 24 21 30 500097 99 69 70 71 81 120 127 100 115 93 92 10000 94 93 92 91 78 84 114 129115 120 89 98 Supernatant dilution (fold) 2B12 30A1 26D5 23D7 23H2 22G3100 89 90 21 24 83 84 31 28 142 176 16 16 1000 64 70 50 57 49 51 54 5388 134 20 23 5000 128 127 126 139 92 96 112 131 117 120 89 99 10000 128133 133 129 95 84 124 148 111 136 86 101 Supernatant dilution (fold)21D4 2G11 4B6 9E2 20B10 100 9 10 22 22 24 31 102 104 6 5 1000 16 17 2927 46 49 77 91 0 4 5000 107 100 66 72 88 94 137 152 25 31 10000 108 11266 72 102 109 137 143 52 58 Supernatant dilution Unrelated (fold) 14G612H12 11D1 26H12 hybridoma 100 18 17 26 23 117 101 106 119 156 185 100027 27 46 56 127 118 124 115 136 144 5000 105 83 145 137 109 99 137 166126 132 10000 113 109 122 145 91 98 151 167 138 137

Example 3 Hybridoma Sub-Cloning

Hybridoma cell lines were subcloned using standard limiting dilutiontechniques. Cells were diluted to a concentration of 50, 5, or 0.5cells/mL. 200 ul of the diluted cell suspensions were plated into 96well tissue culture plates. The plates were incubated at 37° C. with 5%CO₂ and −90% relative humidity. The growth was visually checked at day 7for macroscopic colonies. Supernatants from wells were screened forantibody production when colony growth was visible. Table 8 shows thesubclone identification nomenclature and monikers. This data indicatesthat several anti-NGF antibodies could be isolated from a clonalpopulation of cells.

TABLE 8 Hybridoma Subclone Identification and Monikers HybridomaSupernatant Subcloned Name Hybridoma Name Monker Lot # 14G6ML129-14G6.3H3 PR-1254970 1764671 2G11 ML129-2G11.3B1 PR-1254971 173467320B10 ML129-20B10.3F4 PR-1254972 1734675 2B12 ML129-2B12.5G9 PR-12549731734676 17G6 ML129-17G6.3E7 PR-1254974 1734677 21D4 ML129-21D4.4A11PR-1254977 1734678 4B6 ML129-4B6.4H3 PR-1254978 1734679 22G3ML129-22G3.3F3 PR-1254979 1734680 23F1 ML129-23F1.4G3 PR-1254980 173468114A9 ML130-14A9.5B12 PR-1254981 1734682 3F7 ML130-3F7.4A8 PR-12549821734683

Example 4 Scale Up and Purification of Monoclonal Antibodies

Subcloned hybridoma cell lines were expanded into Hybridoma SFM(Invitrogen catalog #12045) with 5% Low IgG Fetal bovine serum(Invitrogen catalog #16250-078). Supernatants were harvested,centrifuged and filtered to remove cellular debris, and concentrated.Antibodies were mixed with Pierce binding buffer A (Thermo, catalog#21001) in a 1 ratio. The antibodies were loaded onto a recombinantProtein A sepharose (GE Healthcare, catalog #17-1279-04) chromatographycolumn, eluted using Pierce elution buffer (Thermo, catalog #21004),neutralized using 2M Tris pH 7.5, and then dialyzed into PBS. This workallowed the isolation of anti-NGF mAbs for characterization studies.

Example 5 Cloning of Canine NGF

The coding region of canine NGF was amplified from canine universal cDNA(Biochain Institute, catalog #4734565) using primers of SEQ ID NO: 45and SEQ ID NO: 46 or primers of SEQ ID NO: 47 and SEQ ID NO: 48 andcloned into a mammalian or bacterial expression vector, respectively.The PCR reactions were set up as recommended by the manufacturer(Novagen, KOD Hot Start Master Mix, catalog #71842-3). The mammalianclone was made as a C-terminal 6-His fusion protein by ligating the PCRproduct with pTT6 vector (Abbott) at the KpnI/XbaI restriction sites.The bacterial clone was made with the pro-NGF sequence using themammalian clone as a template and ligated with pET15B (Novagen) at theNdeI/XhoI restriction sites. The DNA sequence and amino acid sequence ofthe canine NGF isolated are listed as SEQ ID NO: 49 and SEQ ID NO: 50,respectively. This work allowed expression of canine NGF protein forpurification.

Example 6 Expression of Canine NGF

The canine NGF clone in the bacterial expression vector was grown at 37°C. in overnight express auto inducing Terrific Broth (Novagen) inRosetta2 (DE3) E. coli host (EMD Biosciences) in 2 L non-baffled flasks.The cells were centrifuged down and the cell paste was resuspended in100 mL of lysis buffer (25 mM Tris, 300 mM NaCl, 10% glycerol, 0.1%Triton X 100 pH 8.0) with lysonase and sonicated for 2 min on ice. Thesample was centrifuged at 15000 RPM and the pellet was solubilized in 50mL of 25 mM Tris, 6 M GdHCl pH 8.0. The sample was centrifuged at 15000rpm for 30 min and the supernatant was loaded on to a 10 ml IMAC resin.

A. IMAC Chromatography

A 10 ml GE-Ni FF column was prepared. Buffer A: 25 mM Tris, 6 M GdHCl pH8.0, Buffer B: A+500 mM Imidazole. The resin was equilibrated and loadedwith recirculation to allow for complete binding (˜10 passes) overnightat 4° C. The column was washed with Buffer A. Batch elution was carriedout with 40 ml Buffer A, followed by 30 ml Buffer A. 5 ml fractions werecollected and pooled.

B. Refolding by Rapid Dilution

The pooled fraction was reduced by adding 50 mM DTT, and EDTA was addedto 10 mM, and incubated for 1 h at RT. The sample was acidified byadding 6M HC1 to pH 4.0 and dialyzed into 6M GdHCl pH 5.0 to removeexcess DTT. Refolding was performed by diluting the reduced/acidifiedsample in 1 L of 100 mM Tris, 1 M Arginine, 5 mM EDTA, 5 mM GSH, and 1mM GSSG pH 9.5 for 4 h at 4° C. The refolded protein was dialyzedagainst 25 mM Tris, 200 mM NaCl, 10% Glycerol pH 8.0. Precipitation wascleared by filtration. The clarified sample was concentrated anddiafiltered into 25 mM Tris, 200 mM NaCl, 10% Glycerol pH 8.0 using a10K membrane.

C. Ni-IMAC

Refolded pro-NGF was loaded on a 5 ml Ni-IMAC. Buffer A: 25 mM Tris, 300mM NaCl, 10% Glycerol pH 8.0. Buffer B: A+500 mM Imidazole. The columnwas washed with Buffer A. 8 ml fractions were collected. Elution wasperformed with a linear gradient 0-100% Buffer B. 5 ml fractions werecollected. Samples of each fraction were mixed with non-reducing NuPageSLB (Invitrogen) and separated on a 4-12% NuPAGE Novex Bis-Tris Midi gelfor analysis. Fractions containing protein were pooled and dialyzedagainst 20 mM Na Phosphate, 50 mM NaCl, 10% glycerol pH 7.4.

D. Trypsin Digestion

Pro-β NGF was mixed with trypsin in resuspension buffer and incubated onice for 30 min. Immobilized inhibitor was added and incubated for 15 minand then filtered.

E. Sepharose Cation Exchange Chromatography

The sample was loaded on a 5 ml SP Sepharose high performancechromatography column (GE Healthcare). Buffer A: 20 mM Na Phosphate, 50mM NaCl, 10% Glycerol pH 7.4, Buffer B: A+1 M NaCl. The column waswashed with Buffer A. Elution was performed with linear gradient 0-100%Buffer B. 5 ml fractions were collected. The fractions were separated ona 4-12% Criterion XT Bis-Tris Midi gel for analysis. Fractionscontaining protein were pooled, dialyzed in PBS pH 7.4, andconcentrated.

This work resulted in the production of several milligrams of purifiedcanine NGF for characterization studies and for studies of anti-NGFcanine antibodies.

Example 7 Characterization of Subcloned and Purified HybridomaAntibodies A. Canine NGF Direct Binding ELISA

To determine if purified mouse anti-NGF mAbs bind to canine β NGF, ELISAplates were coated with 50 μl/well of canine NGF (Abbott Laboratories)at 1 μg/ml in PBS and incubated over night at 4° C. The plates werewashed three times with PBS+Tween buffer. The plates were blocked with200 μl/well of 2% milk in PBS for 1 hour at room temperature. The plateswere washed three times as above. Purified antibodies were diluted to0.4, 2, or 10 μg/ml. 50 μl of each concentration of purified antibodywas added to the plates. The plates were incubated for 1 hour at roomtemperature. The plates were washed. 50 μl of a 5000-fold diluted goatanti-mouse IgG Fc-HRP (Thermo, catalog #31439) was added. The plateswere incubated for 1 10 hour at room temperature. 50 μl of TMB(Invitrogen, catalog #00-2023) was added and the reaction was stoppedusing 2N H₂SO₄ (VWR, catalog #BDH3500-1). The absorbance at 450 nm wasread on a Spectromax 2E plate reader (Molecular Devices). The resultsare shown in Table 9, and the numerical value indicates binding of mouseanti-NGF antibodies to canine β NGF.

TABLE 9 Canine NGF Direct Binding ELISA Data Using Purified Anti-NGFmAbs μg/ml PR- PR- PR- PR- PR- PR- PR- PR- PR- PR- PR- Mab 12549701254971 1254972 1254973 1254974 1254977 1254978 1254979 1254980 12549811254982 10 0.530 0.497 0.154 0.905 0.552 0.552 0.579 0.683 0.491 0.6100.208 2 0.342 0.324 0.091 0.836 0.383 0.414 0.458 0.566 0.334 0.4580.142 0.4 0.176 0.165 0.071 0.769 0.209 0.223 0.253 0.313 0.168 0.2290.095

B. TF-1 Cell Proliferation Potency Assay

TF-1 is a human erythroleukaemic cell line that expresses human TrkA andproliferates in response to recombinant β NGF. To determine if purifiedanti-NGF mAbs blocked NGF-induced proliferation, TF-1 cells(ATCC#CRL-2003) were maintained at 37° C. and 5% C0₂ in RPMI (Gibco, cat#1 1875-093) media containing recombinant human GM-CSF at 2 ng/mL (R&DSystems, cat #215-GM) and fetal bovine serum (FBS, Hyclone, cat #SH30070.03). GM-CSF and FBS was removed 24 hours before the assay. On dayone of the assay each anti-NGF mAb was titrated (concentrations rangingfrom 33.3 nM to 1.7 fM) and added to a fixed concentration ofrecombinant canine NGF (70 pM) and TF-1 cells (2.5×10⁻⁴ cells/well) inRPMI+4% FBS for 72 hours. Cell proliferation was measured using CellTiter-glo (Promega, cat #G7571). The IC₅0 values of each anti-NGF mAb oncanine NGF-induced TF-1 cell proliferation is shown in Table 10 and thedata shows that in the presence of 70 pM canine NGF, most of theanti-NGF antibodies display sub-nM potencies, and some display potenciesof less than 50 pM.

TABLE 10 Potency of Mouse Anti-NGF Antibodies on Canine NGF-induced TF-1Cell Proliferation Moniker Lot IC50 (nM) PR-1254970 1764671 0.662PR-1254971 1734673 1.088 PR-1254972 1734675 0.303 PR-1254973 17346760.039 PR-1254974 1734677 0.230 PR-1254977 1734678 0.217 PR-12549781734679 0.978 PR-1254979 1734680 0.288 PR-1254980 1734681 0.343PR-1254981 1734682 0.046 PR-1254982 1734683 0.025C. SureFire Cellular pERK and PathHunter Assays

To determine if purified mouse anti-NGF mAbs blocked canine NGF-inducedcellular responses, purified antibodies were characterized by titrationin the SureFire cellular pERK (using 128 pM canine β NGF in each testwell) and PathHunter assays (using 441 pM canine NGF in each test well)as described in Example 2 Sections C and D. The IC₅o of each anti-NGFmAb on canine β NGF-induced cellular responses is summarized in Table 11and the data shows that in the presence of 128 pM canine NGF all theanti-NGF antibodies display sub-nM potencies, and some display potenciesof less than 50 pM (pERK assay). Also, in the presence of 441 pM canineNGF, all the anti-NGF antibodies display sub-nM potencies, and somedisplay potencies of less than 150 pM (PathHunter assay).

TABLE 11 Summary of pERK and Path Hunter Assay Data for PurifiedAnti-NGF mAbs SureFire pERK PathHunter Antibody IC₅₀ (nM) IC₅₀ (nM)PR-1254970 0.02711 0.3346 PR-1254971 0.04750 0.4986 PR-1254972 0.22820.3133 PR-1254973 0.01876 0.1428 PR-1254974 0.01561 0.2464 PR-12549770.01759 0.1810 PR-1254978 0.02466 0.3559 PR-1254979 0.01627 0.2414PR-1254980 0.01371 0.3812 PR-1254981 0.02135 0.2794 PR-1254982 0.0058040.1505

Example 8 Characterization of Purified Anti-NGF Antibodies FollowingHybridoma Subcloning A. Mass Spectrophotometry (MS) and Size ExclusionChromatography (SEC) Analysis on Anti-NGF Antibodies

The mouse anti-NGF mAbs were reduced using 1M DTT and analyzed usingHPLC/MS on a 6224 TOF mass spectrometer and a 1200 HPLC (Agilenttechnologies) using a Vydac C4, IMM×150 mm column (CN#214TP5115, theNest Group) at a flow rate of 50 μl/min. Buffer A: 99.9% HPLC water+0.1%FA+0.01% TFA and buffer B: 99.9% ACN+0.1% FA+0.01% TFA. The LCequilibrium and sample desalting was performed using 5% buffer B for 7min. The separation gradient was performed using 30% to 50% Buffer B for10 min and a washing step was performed at 95% buffer B for 10 mins. TheTOF acquisition parameters were: gas temperature at 350 C and OCT/RF at750V. The mass range was from 600-3200 m/z and the rate specified was1.03 spectra/s. Qualitative analysis software (Agilent) was used todeconvolute antibody molecular weights.

The antibodies were analyzed on Shimadzu LC-10AVP system (ShimadzuScientific). The SEC column used was a Superdex-200 10/300L (GEHealthcare). The flow rate was 0.75 ml/min and UV280 was used to monitorpeaks. The buffer used was Na₂S0₄+92 mM NaP0₄+5 mM NaZ₃, pH 7.0. Thereagent antibody was injected in 10 μl, (10 μg). The gel protein markerson SEC were from Bio-Rad (CN#151-1901). The MS and SEC results aresummarized in Table 12. This data determined the hybridoma-derivedantibodies were highly monomeric following purification. In addition,the molecular weights of the heavy and light chains comprising thehybridoma-derived antibodies were determined.

B. Antibody Isotype Determination

The isotype of the anti-NGF mAbs was determined using the Zymed MouseMonoAb-ID Kit (Invitrogen catalog#90-6550 lot#1407589). The isotypingresults are summarized in Table 12. This data indicates that murineIgG1/k, IgG2a/k, and IgG2b/k mouse antibodies are capable of binding andneutralizing NGF.

TABLE 12 Isotyping, Size Exclusion Chromatography, and Mass SpectrometryAnalysis of Anti-NGF Antibodies Molecular Molecular weight (Dal) weight(Dal) Heavy Hybridoma Name Moniker Lot Isotype % Monomer Light ChainChain ML129-14G6.3H3 PR-1254970 1734671 IgG1 Kappa 96.9 24221.4349479.67 ML129-2G11.3B1 PR-1254971 1734673 IgG1 Kappa 96.8 24156.2649491.69 ML129-20B10.3F4 PR-1254972 1734675 IgG2b Kappa 99.0 24159.3450329.24 ML129-2B12.5G9 PR-1254973 1734676 IgG2b Kappa 99.4 23539.3851102.21 ML129-17G6.3E7 PR-1254974 1734677 IgG1 Kappa 98.8 24221.4349479.45 ML129-21D4.4A11 PR-1254977 1734678 IgG1 Kappa 98.4 24221.4649479.70 ML129-4B6.4H3 PR-1254978 1734679 IgG1 Kappa 96.7 24170.4049533.92 ML129-22G3.3F3 PR-1254979 1734680 IgG2a Kappa 99.0 24221.4250123.17 ML129-23F1.4G3 PR-1254980 1734681 IgG1 Kappa 99.5 24221.4249493.95 ML130-14A9.5B12 PR-1254981 1734682 IgG1 Kappa 99.1 24180.2850241.85 ML130-3F7.4A8 PR-1254982 1734683 IgG1 Kappa 99.4 23708.5450289.13

Example 9 Binding Kinetics of Anti-NGF Antibodies

A biomolecular protein interaction analysis was used to evaluate thebinding kinetics of the interaction between the purified anti-NGFhybridoma antibodies and recombinant canine β NGF. The antibodies werecaptured using a goat anti-mouse IgG FC (10000 RU) surface which wasdirectly immobilized to a CM5 chip using an amine coupling procedureaccording to the manufacturers instructions (Biacore). A sample size of5 μl of antibody at a concentration of 1 μg/ml was captured at 10 μlminute. Recombinant canine NGF was used as the antigen. Canine NGF wasinjected at 75 μl/min (concentration range: 5-0.039 nM) for mouseantibodies. The association rate was monitored for 3.3 minutes and thedissociation rate was monitored for 10 minutes. Aliquots of canine NGFwere also simultaneously injected over a reference reaction CM surfaceto record any nonspecific binding background. The instrument sensitivityfor on-rate is I×IO⁷, such that any on-rate that is faster than I×IO⁷may not be accurately measured; the instrument sensitivity for off-rateis 1×10⁶, such that any off-rate that is slower than 1×10⁻⁶ may not beaccurately measured. Therefore, an on-rate that is faster than 1×10⁷ isrecorded as >1×10⁷ and an off-rate that is slower than 1×10⁻⁶ isrecorded as <1×10⁻⁶ The biomolecular protein interaction analysisresults are summarized in Table 13. This data indicates that theisolated murine anti-NGF mAbs have fast on-rates (from greater than7×10⁶) and slow off-rates (from less than I×IO⁻³). The overall KDs ofthe murine anti-NGF mAbs range from about 300 pM to 0.1 pM demonstratingefficient binding of the purified anti-NGF hybridoma antibodies torecombinant canine β NGF.

TABLE 13 Binding Kinetics of Anti-NGF mAbs to Canine NGF On rate Offrate Overall Antibody (1/Ms) (1/s) affinity(M) PR-1254972 Expt 1 >1 ×10⁷ 3.14 × 10⁻³ <3.14 × 10⁻¹⁰ lot: 1734675 Expt 2 >1 × 10⁷ 3.21 × 10⁻³<3.21 × 10⁻¹⁰ Average >1 × 10⁷ 3.18 × 10⁻³ <3.18 × 10⁻¹⁰ PR-1254973 Expt1 >1 × 10⁷ 1.21 × 10⁻⁴ <1.21 × 10⁻¹¹ lot: 1734676 Expt 2 >1 × 10⁷ 1.38 ×10⁻⁴ <1.38 × 10⁻¹¹ Average >1 × 10⁷ 1.30 × 10⁻⁴ <1.30 × 10⁻¹¹ PR-1254977Expt 1 >1 × 10⁷ 1.39 × 10⁻⁴ <1.39 × 10⁻¹¹ lot: 1734678 Expt 2 >1 × 10⁷1.60 × 10⁻⁴  <1.6 × 10⁻¹¹ Average >1 × 10⁷ 1.50 × 10⁻⁴  <1.5 × 10⁻¹¹PR-1254980 Expt 1 >1 × 10⁷ 2.37 × 10⁻⁴ <2.37 × 10⁻¹¹ lot: 1734681 Expt2 >1 × 10⁷ 2.25 × 10⁻⁴ <2.25 × 10⁻¹¹ Average >1 × 10⁷ 2.31 × 10⁻⁴ <2.31× 10⁻¹¹ PR-1254981 Expt 1 8.67 × 10⁶  1.27 × 10⁻⁴  1.47 × 10⁻¹¹ lot:1734682 Expt 2 7.48 × 10⁶  1.40 × 10⁻⁴  1.87 × 10⁻¹¹ Average 8.08 × 10⁶ 1.34 × 10⁻⁴  1.67 × 10⁻¹¹ PR-1254982 Expt 1 >1 × 10⁷   >1 × 10⁻⁶   >1 ×10⁻¹³ lot: 1734683 Expt 2 >1 × 10⁷   >1 × 10⁻⁶   >1 × 10⁻¹³ Average >1 ×10⁷  >1e × 10⁻⁶   >1 × 10⁻¹³

Example 10 Method for Identifying Anti-NGF Antibody Sequences fromHybridomas by Cloning and Sequencing

To identify the nucleotide and amino acid sequence of the six subclonedhybridoma mAbs shown in Table 13, the RNA from individual hybridomacultures was extracted with Qiagen RNeasy kit (Qiagen, cat #74104). RNAwas reverse-transcribed and cDNA antibody sequences amplified using theQiagen One-Step RT-PCR kit (Qiagen, catalog #210212). Forward primerswere degenerate and designed to anneal to the variable regions (heavychain primers: 1HA, 1HB, 1HC, 1HD, 1HE, 1HF; and light chain primers:1LA, 1LB, 1LC, 1LD, 1LE, 1LF, 1LG) (EMD4 Biosciences catalog #69896).Reverse primers were also degenerate and made to constant regions ofgamma (heavy chains) and kappa (light chains). PCR products ofapproximately 400-450 base pairs were gel isolated and purified withQiagen Gel Extraction kit (Qiagen, cat #28706).

Purified PCR products were cloned into TOPO TA cloning vectors(Invitrogen, cat # K4500-01SC). Each topoisomerase reaction mixture wasused to transform TOP 10 chemically competent bacteria and plated on LBplates with 75 μg/ml Ampicillin and 60 μl 2% Bluo-Gal (Invitrogen, cat#15519-028). Isolated colonies were picked from the LB plate toinoculate 20 μl LB broth/100 μg/ml carbenicillin. One μl of thismini-culture was used in a PCR reaction with MI 3 forward and reverseprimers to amplify the insert in the TOPO vector. PCR products wereseparated on 2% agarose gels; samples indicating an appropriately-sizedinsert in the vector were sequenced using an Applied Biosystems model3730S DNA sequencer. DNA sequences derived from the identification ofall murine mAb heavy and light chain variable domains were translatedinto protein sequence and are shown in FIG. 1 to FIG. 24.

Example 11 Homology Modeling of Murine Anti-NGF Antibodies

The sequences of the heavy and light chain variable regions of eachanti-NGF antibody were imported into InsightII (Accelrys, San Diego,Calif.). Each sequence was used as a template for BLAST to find thex-ray crystal structures from the Protein Data Bank (www.rcsb.org) whichwere closest in identity. One structure for each of the heavy and lightchains was selected based both on percent identity and on matching theexact length of all CDR loops. The sequences of each template and eachquery sequence were aligned and standard homology modeling techniquesused to construct homology models of each chain. The complex of bothmodeled chains was then minimized for 50 cycles of restrained (500Kcal/Angstrom for all heavy atoms) conjugate gradient minimization usingthe CVFF force field in the DISCOVER program (Accelrys, San Diego,Calif.).

The likelihood that a given framework residue would impact the bindingproperties of the antibody depends on its proximity to the CDR residues.Therefore, using the model structures, residues that fell within 5 A ofany CDR atom was identified as most important and were recommended to becandidates for retention of the murine residue in the caninized antibodysequences. A change in nucleotide(s) in a mutant gene that restores theoriginal sequence and hence the original phenotype is often referred toas a back mutation. Therefore, we refer to residues that are candidatesfor retention of the murine residue in the caninized antibody sequencesas backmutations.

Example 12 Identification of Canine Heavy and Light Chain AntibodySequences from Canine PBMCs

To identify canine Ig heavy and lambda light chain antibody variabledomain amino acid sequences, RNA was isolated from mongrel canineperipheral blood mononuclear cells (PBMCs) using an RNEasy kit (Qiagen#74104). Canine PBMC mRNA was reverse transcribed (RT) with SuperscriptIII reverse transcriptase (Invitrogen catalog #18080-093) and cDNAs wereamplified using the 5′ RACE System (Rapid Amplification of cDNA Ends)(Invitrogen #18374-058). RT and PCR primers (RK323, RK324, RK122, LG010,LG011, LG012) are described in patent publication number: U.S. Pat. No.7,261,890 B2 entitled Methods for Using Canine Immunoglobulin VariableDomains and Caninized Antibodies). Primers RK323 and RK324 were used forcanine IgG reverse transcription followed by nested PCR with RK326 andthe Abridged Anchor Primer (AAP) (Invitrogen). LG011 was used for caninelambda light chain RT PCR, followed by nested PCR with LG010 and LG012and AAP.

The resulting PCR products were separated by agarose gelelectrophoresis. The 600 base pair (canine lambda and kappa lightchains) and 800 base pair (canine Ig heavy chain) PCR products werepurified from the agarose using a Gel Extraction kit (Qiagen #28706) andcloned into the TA site of the pCR2.1 TOPO vectors using the TOPO-TACloning system (Invitrogen #K4500-01 SC).

Transformed TOP 10 bacteria were selected and plasmid DNA was isolatedusing Qiaprep Spin Mini-Prep Kit (Qiagen #27104). Plasmid DNA from 25heavy chain, 38 kappa light chain and 23 lambda light chain colonies wassequenced to identify the nucleotide and corresponding amino acidsequences. Complete variable domain sequence data were obtained from 25heavy chain, 38 kappa light chain and 19 lambda light chain clones.Variable domain sequence data including the leader peptide (whenidentified) are shown in Tables 14, 15 and 16. All derived heavy chainand light chain sequence are unique compared to those disclosed inpatent publication number: U.S. Pat. No. 7,261,890 B2.

TABLE 14 Canine Heavy Chain Variable Domain SequencesDerived from Canine PBMC RNA Name Sequence Ca-1005EVQLEESGGDLVKPGGSLRLSCVASGFSIGSYGMSWVRQSPGKGLQWVAWIKYDGSRTFYADAVKGRFTISRDNAKNTLFLQMNSLRAEDTAVYFCVKGPNSSWLPST YFASWGQGTLVTVSS (SEQ ID NO: 178)Ca-2301 EMQLVESGGDLVRPGGSLRLSCVASGFTFSTYGMTWVRQSPGKGLQWVATIGPGGRNTYYADAVKGRFTISRDDAENTLFLQMNSLRAEDTAVYYCAQAFDATYYTSF DCWGRGSLVAVSS (SEQ ID NO: 86)Ca-2302 MESVLSWVFLVALLQGIQGEIRLVESGGDLVKPGGSLRLSCVASGFIFGNYDMSWVRQAPGKGLQWVAAVRYDGSSTYYSDAVKGRITISRDDPGNTVYLQLDSLRAEDTATYYCVRGGYYSSSFYIGGAFGHWGPGTLITVSS (SEQ ID NO: 87) Ca-2303MECVLGWVFLVAILRGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTFSDYYMSWIRQAPGKGLQWVADISDGGDGTGYAGAVKGRFTVSRENVKNTLYLQMNDLRAE DTAIYYCTKAREMYGYRDFDSWGPGTLVTVSS(SEQ ID NO: 88) Ca-2304 MESVLGLVALLTILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTFSNYYMTWVRQAPGKGLEWVGYIHNGGTYTYYADAVKGRFTISRDDAKNTLYLEMNSLRAE DTAVYYCGKMIFDYWGQGTLVTVSS(SEQ ID NO: 89) Ca-2305 MESALSWVFLVTILKGVQGEVLLVESGGDLVKPGGSLRLSCLTSGFTFNTYDWGWVRQAPGKGLQWIAYIKKGGSDVRYADAVKGRFTISRDDAKNTLYLQMNSLRAE DTAVYYCARSAWDSFDYWGQGTLVTVSS(SEQ ID NO: 90) Ca-2306 MESVFCWVFLVAILKGVRGVQGEVQLVESGGDLVKPAGSLRLSCVASGFTFTDYSMNWVRQAPGKGLQWVATISNDGTSTDYTDAVKGRFTVSRDSARNTVYLQMTSL RADDTATYYCVSRHSYSLLADYWGQGTLVTVSS(SEQ ID NO: 91) Ca-2307 MQMPWSLLCLLAAPLGVLSEVTLQESGPGLVKPSQTLSLTCAVSGGSVIRNYYWHWIRQRPGRGLEWMGCWSETTYYSPAFRGRISITIDAATDQFSLHLNSMTTDDT AVYYCARALYPTSSWYDGMDYWGHGASVVVSS(SEQ ID NO: 92) Ca-2308 EVQLVESGGDLVKPGGSLRLSCESSGFIFSQYAMNWVRQAPGKGLQWVAYIGGAGFITYHADDVKGRFTISRDNAKNTLYLQMNSLTINDTAVYYCVRSNSRIPDYWG QGTLVAVSS (SEQ ID NO: 93) Ca-2309MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTFSSVYMSWVRQAPGLQWVARITTDGTDTFYADAVKGRFTISRDNVKNMLYLEMNSLRAEDT AIYYCGDPWQPAYPDLWGQGTMVTVSS(SEQ ID NO: 94) Ca-2310 MESVLCWVFLVAILKGVQGEVHLVESGGDLVKPGGTLRLSCVASGFTFSQYDMSWVRQSPGKGLQWVALSRYHGGGTYYADAVKGRFTISRDNAKNMLYLQMNSLRAE DTAVYYCVKEGSRWDLRGDYDYWGQGTLVTVSS(SEQ ID NO: 95) Ca-2311 MQMPWSLLCLLAAPLGVLSELTLQESGPGLVKPSQTLSLTCVVSGGSVTSSHYWNWIRQRPGRGLEWMGYWTGNVNYNPAFQGRISIIGDAAKNQFSLHLSSMTTDDT AVYYCARCGIVAPGFLPIGDFDFWGQGTLVTVSS(SEQ ID NO: 96) Ca-2312 MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFSFSNYFMFWGRQAPGKGLQWVARIRSDGGSTYYADAVKGRFTISRDNARNTLYLQMNSLRAE DTATYYCAKADIIKLPEYRGQGTLVTVSS(SEQ ID NO: 97) Ca-2401 ESVLGWIFLATILKGVQGEVQLVESGGDLVKPGGSLRLSCVGSGFTFSSSWMNWVRQAPGKGLQWIAEISGTGSSTNYADAVKGRFTISRDNDKNTLYLQMNSLRAED TAMYYCARAAYYGNYRNDLDYWGQGTLVTVSS(SEQ ID NO: 98) Ca-2402 KPAGSLRLSCVASGFTFSSHSVTWVRQAPGKGLQFVAGITSGGNNRYYTDAVRGRFTLSRDNAKNTVYLQMNSLRAEDTAMYFCALGSYEWLSGEFDYWGQGTLVTVS S (SEQ ID NO: 99) Ca-2403MESVFCWVFLVAILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTLNNYFMYWVRQAPGKGLQWVARLNSNGDSTFYADAVKGRFTISRDNAKNTLYLQMNSLRAE DTSMYYCAKDLIYGYTLWGQGTLVTVSS(SEQ ID NO: 100) Ca-2404 MASVLSWVFLVAIVKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFIFNKYEVYWVRQAPGKGLEWVARILESGNPTYYAEAVEGRFTISRDNAKNMAYLQMNSLRAD DTAVYYCATPSVSSTVAIDYWGQGALVTVSS(SEQ ID NO: 101) Ca-2405 MQMPWSLLCLLATPLGVLSELTLQESGPGLVKPSQTLSLTCVVSRGSVTSDYYWNWIRQRPGRGLEWMGHWIGSTAYNPAFQGRISITADTAKNQLSLQLRSMTTEDT AVYFCARGSSWTPSGDSWGQGTLVTVSS(SEQ ID NO: 102) Ca-2406 MASVLKLGFSCRYCKKVSRVRCNXVESGGDLVKPGGSLRLSCVASGFIFNKYEVYWVRQAPGKGLEWVARILESGNPTYYAEAVEGRFTISRDNAKNMAYLQMNSLRA DDTAVYYCATPSVSSTVAIDYWGQGALVTVSS(SEQ ID NO: 103) Ca-2407 MDCSWRIFFLLALATGVHSEVQLVQSAAEVKKPGASVKVSCKTSGYTLTDYYIHWVQQAPGTGLHWMGWIDPEXGTTDYAQKFQGXVTLTADTSTNTAYMELSGLRAE DTAVYYCARFPRSLDYGSFPFDYWGQGTLVTVSS(SEQ ID NO: 104) Ca-2408 MESVLCWVFLVAILKGVQGEVRLVESGGDLVKPGGSLRLSCVASGFTFRNYGMSWVRQRPGKGLQWVAAIRSDGVTYYADDLKVRFTVSRDDARNTLYLQLNSLGAED TAVYYCAKAPWGLYDAWGQGTLVTVSS(SEQ ID NO: 105) Ca-2409 MESVLSWVFLVAILQGVQGEVQVVESGGDLVKPAGSLRLSCVASGYSISTYTMTWVRQVPGKGLQLVAGINGDGSSTYYTDAVKGRFTISRDNARNTVYLQMNSLRAE DTAMYYCLGEYSWFYYWGQGTLVTVSS(SEQ ID NO: 106) Ca-2410 MQMPWSLLCLLAAPLGVLSELTLQESGPRLVKPSQTLSLTCAVSGGSVTTTSYWSWIRQRPGRGLEWVGYWTGTTNYSPAFQGRISISADTAKNQFSLHLSSVTTEDT ALYFCASKSASTSWYFSLFESWGQGTLVTVSS(SEQ ID NO: 107) Ca-2411 MESVLGLVFLLTILKGVQGEVQLVESGGDLVKPGGSLRLSCVASGFTFSSYSMSWVRQAPGKGLQWVGYIDNGGTSTYYADAVKGRFTISRDNAKNTLYLQMNSLRAE DTAVYYCGRGSYGMEYWGHGTSLFVSS(SEQ ID NO: 108) Ca-2412 MESVLGLLFLVAILKGVQGEIQLVESGGDLLKPGGSLRLSCVASGFTFSGSDMNWIRQAPGKGLQWVAHITHEGIGTSYVGSVKGRFTISRDNAKNTLYLQMNDLRAE DTAMYYCAYSPWNYYSFDSWGQGTLVTVSS(SEQ ID NO: 109)

TABLE 15 Canine Lambda Light Chain VariableDomain Sequences Derived from Canine PBMC RNA Name Sequence Ca-1001MTSTMAWSPLLLTLLTHCTVSWAQTVLTQSPSVSAVLGRRVTISCTGSDTNIGSHRDVQWYQLVPGKSPKTLIYGTDNRPSGIPVRFSGSKSGNSGTLTITGIQAEDEADYYCQSYDDDLSMNVFGGGTHLTVLG (SEQ ID NO: 110) Ca-1002MDWVPFYILPFIFSTGFCALPVLTQPTNASASLEESVKLTCTLSSEHSNYIVRWYQQQPGKAPRYLMYVRSDGSYKRGDGIPSRFSGSSSGADRYLTISNIKSEDEDDYYYCGADYTISGQYGSVFGGGTHLTVLG (SEQ ID NO: 111) Ca-1003LWISGGSALGTPTMAWTHLLLPVLTLCTGSVASSVLTQPPSVSVSLGQTA TISCSGESLSKYYAQWFQQKAGQVP VLVIYKDTERPSGIPDRFSGSSSGNTHTLTISRARAEDEADYYCESEVSTGTYCVRRRHPSNRPRSAQGLPLGHTL PALL (SEQ ID NO: 204) Ca-1006MTSTMAWSPLLLTLLTHCTGSWAQSVLTQPASLSGSLGQRVTISCTGSSSNIGGYSVNWLQQLPGTGPRTIIYNNSNRPSGVPDRFSGSRSGTTATLTISGLQAEDEADYYCSTWDSNLRTIVFGGGTHLTVLG (SEQ ID NO: 112) Ca-1007MTSTMDWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLGQRVTISCTGSTSNLGTYNVGWLQQVPGTGPRTVIYTNIYRPSGVPDRFSGSESGSTATLTISDLQAEDEAEYYCTAWDSSLNAYVFGSGTQLTVLG (SEQ ID NO: 113) Ca-1008MTSNMAWCPFLLTLLAYCTGSWAQSVLTQPTSVSGSLGQRVTISCSGSTNNIGIVGASWYQQLPGKAPKLLVYSDGDRPSGVPDRFSGSNSGNSDTLTITGLQAEDEADYYCQSFDTTLDAAVFGGGTHLTVLG (SEQ ID NO: 114) Ca-1009MTSTMAWSPLLLTLLAHCTVSWAQAVLTQPPSVSAALGQRVTISCTGSDTNIGSGYEVHWYRQVPGKSPAIIIYGNSNRPSGVPVRFSGSKSGSTATLTITGIEAEDEADYHCQSYDGNLDGGVFGGGTHLTVLG (SEQ ID NO: 115) Ca-1010MTSTMGWFPLILTLLAHCAGSWAQSVLTQPASVSGSLGQRVTISCTGSSPNVGYGDFVAWYQQVPGTSPRTLIYNTRSRPSGVPDRFSASRSGNTATLTISGLQAEDEADYYCSSYDNTLIGIVFGGGTHLTVLG (SEQ ID NO: 116) Ca-1011MTSTMGWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLGQRVTITCTGSSSNIGRANVAWFQQVPGTGPRTVIYTSVKRPSGVPDRFSGSKSGSTATLTISGLQAEDEADYYCSSWDNSLDAGVFGGGTHLTVLG (SEQ ID NO: 117) Ca-1012MTSTMGWFPLLLTLLAHSTGSWAQSVLTQPASVSGSLGQRVTITCTGGTSNIGRGFVSWFQQVPGIGPKILIFDAYRRPSGVPDRFSGSRSGNTATLTISGLQAEDEADYYCAVYDSRLDVGVFGSGSQLTVLS (SEQ ID NO: 118) Ca-1202MTSNMAWCPFLLTLLTYCTGSWARSVLTQPASVSGSPGQKVTIYCSGTMSDIGVLGANWYQQLPGKAPKLLVDNDGDRPSGVPDRFSASKSGHSDTLTITGLQPEDEGDYYCQSFDSSLDAAIFGEGTHLTVLG (SEQ ID NO: 119) Ca-1203SVASYVLTQSPSQNVTLRQAAHITCEGHNIGTKSVHWYQQKQGQAPVLIIYDDKSRPSGIPERFSGANSGNTATLTISGALAEDEADYYCLVWDSSAIWVFGEGTHLTVLG (SEQ ID NO: 120) Ca-1204MTSTMAWSPLLLTLLAHFTGSWAQSVLTQPTSVSGSLGQRVTISCTASSSNIDRDYVAWYQQLPGTRPRALIYANSNRPSGVPDRFSGSKSGSTATLTISGLQAEDEADYYCSTWDNSLTYVFGSGTQLTVLG (SEQ ID NO: 121) Ca-1205SVASYVLTQVPSVSVNLGKTATITCEGDNVGEKYTHWYQQEYGQAPVLIIYEDSRRPSGIPERFSGSNSGNTATLTISGARAEDETDYYCQVWDDSGNVFGGGTHLTVLG (SEQ ID NO: 122) Ca-1206MTSTMGWFPLILTLLAHCAGSWAQSVLTQPASVSGSLGQRVTISCTGSDSNVGYGDSIAYGDSVAWYQQVPGTSPRTLIYDVTSRPSGVPDRFSGSRSGTTATLTISGLQAEDE ADYYCSSFDKTLNGLIVGGGTHLTVLG(SEQ ID NO: 123) Ca-1207 MTSNMAWSPLLLTLLAYCTGSWAQSALTQPTSVSGSLGQRVSISCSGGIHNIGSVGATWYQQLPGKAPKLLVSSDGDRPSGIPDRFSGSRSGNSVTLTITGLQAEDEAEYYCQSFDSTLGVHVVFGGGTHLTVLG (SEQ ID NO: 124) Ca-1208LCSAVGPPKTESVMTSTMGWSPLLLTLLAHCTGSWAQSVLTQPASVSGSLGQRVTIPCTGSSSNIDRYNVAWFQQL PGTGPKPSSIVLLTDPQGSLIDSLAPSQAA(SEQ ID NO: 205) Ca-1209 MTSTMAWFPLLLTLLAHYTGSWARSDLTQPASVSGSLGQRITISCTGSSSNIGRNYVGWYQQLPGRGPRTVVYGINSRPSGVPDRFSGSKSGSTVTLTISGLQAEDEADYYCSTWDDSLSVVVFGGGTHLTVLG (SEQ ID NO: 125) Ca-1210MTSTMGWSPLLLTLTHWTGSWAQSVLSQPASMSGSLGLRITICCTGKNSNINNSYVDWNQPLAGTGPRTVIHDDGDRPSGVPDQFSGSKSGNTATLTISRLQAEDEADYNGASFETSFNAVFGGGTHVTVLG (SEQ ID NO: 126)

TABLE 16 Canine Kappa Light Chain Variable DomainSequences Derived from Canine PBMC RNA Ca Ka016-A1LSWLRQKPGHSPQRLIHQVSSRDPGVPDRFSGS GSGTDFTLTISRVEADDGGVYYCGQGSQSIPTFGQGTKVEIKR (SEQ. ID NO. 127) Ca Ka016-A2MRFPSQLLGLLMLWIPGSAGDIVMTQTPLSLSV SPGEPASISCKASQSLLHSKGNTYLYWFRQKPGQSPQRLIYKVSNRDPGVPDRFSGSGSGTDFTLR ISRVETDDAGVYYCGQVIQDPWTFGVGTKLELKR (SEQ. ID NO. 128) Ca Ka016-A3 MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGETASISCRASQTLLYSNGKNYLFWYRQKPG QSPQRLIDLASNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCGQGMEIPWTFGAGTKVELK R (SEQ. ID NO. 129) Ca Ka016-A4MKFPSLLLGLLMLWIPGSTGEAVMTQTPLSLAV TPGEVATISCRASQSLLHSDGKSYLNWYLQKPG QTPRPLIYEASKRFSGVSDRFSGSGSGTDFTLK INRVEAEDVGVYYCQQSLHFPPTFGPGTKVELKR (SEQ. ID NO. 130) Ca Ka016-A5 PDRFSGSGSGTDFTLTISRVEADDAGIYYCGQATQTPPTFGAGTKLDLKR (SEQ. ID NO. 131) Ca Ka016-A6MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV RPGESASISCKASQSLLHSGGGTYLNWFRQRPG QSPQRLIYEVSKRDTGVPDRFSGSGSGTDFTLR ITRVEADDTGIYYCGQNTQLPLTFGQGTKVEIKR (SEQ. ID NO. 132) Ca Ka016-A7 MRFPSQLLGLLMLWIPGSTGDIVMTQTPLSLSVSPGEPASISCKASQSLLHSNGNTYLFWLROKPG  QSPQRLTYRVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCGQRVRSPWTFGAGTKVEVK R (SEQ. ID NO. 133) Ca Ka016-A8MRFPSQLLGLLMLWIPGSAGDIVMTOTPLSLSV SPGEPASISCKASQSLLHSNGNTYLYWFRQKPG QSPQRLIYKVSKRDPGVPDRFSGSGSGTDFTLR ISRVETDDAGVYYCGQVIQDPWTFGVGTKLELKR (SEQ. ID NO. 134) Ca Ka016-A9 MRFPSQLLGLLMLWIPGSSGDVVMAQTPLSLSVSPGETASISCRASQSLLHSNGNTFLFWFRQKPG  QSPQRLINFLSNRDPGVPDRFSGSGSGTDFTLRINRVEADDAGLYYCGQGLQAPLTFGQGTKLEIK R (SEQ. ID NO. 135) Ca Ka016-A10MRFPSQLLGLLMLWIPGSNGDDVLTQTPLSLSV RPGETVSILCKASESLLHSDGNTYLSWVRQKAG QSPQRLMYRVSDRDTGVPDRFSGSGSGTDFTLT ISGVEADDAGIYYCGQATHYPLEFGQGTRVEIKR (SEQ. ID NO. 136) Ca Ka016-A11 LMLWIPGSTGEIVLTQTPLSLSVSPGEPASISCKASQSLLHPNGVTYLYWFRQKPGQSPQRLIYKV SNRDPGVPDRFSGSGSEIDFTLIISRVEADDGGIYYCGQGIQNPFTFGQGTKLEIKR (SEQ. ID NO. 137) Ca Ka016-A12MRFPSQLLGLLMLWIPGSIGDIVMTQTPLSLSV SPGESASISCKASQSLLHSNGNTYLYWFRQKPGHSPQRLIHQVSSRDPGVPDRFSGSGSGTDFTLR ISRVEADDAGLYYCGQGTQFPFTFGQGTKVEIKR (SEQ. ID NO. 138) Ca Ka016-B1 MRFPSQLLGLLMLWIPGSIGDIVMTQTPLSLSVSPGESASISCKASQSLLHSNGNTYLYWFRQKPG  HSPQRLIHQVSSRDPOVPDRFSGSGSGTDFTLRISRVEADDAGLYYCGQGTQFPFTFGQGTKVEIK R (SEQ. ID NO. 139) Ca Ka016-B2MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSV SPGETASISCRASQSLLHSNGNTYSFWFRQKPG QSPQRLINLVSSRGPGVPDRFSGSGSGTDFTLI ISRVEADDAGVYYCGHGKEAPYTFSQGTKLEIKR (SEQ. ID NO. 140) Ca Ka016-B3 MRFPSQLLGLLMLWIPGSVGDIVMTQSPMSLSVGPGESASMSCKANQSLLYSDGITYLSWFLQRPG  QSPQRLIYEVSKRDTGVPGRFIGSGAGTDFTLRISRVEADDAGVYYCGQALQFPLTFSQGAKLEIE R (SEQ. ID NO. 141) Ca Ka016-B4MRFPSQLLGLLMLWIPGSSGDVVMTQTPLSLSV RPGETASISCRASQSLLHSSGITKLFWYRQKPG QSPQRLVYWVSKRDPGVPDRFTGSGSGTDFTLR ISRLEADDAGIYYCGHAIGFPLTFGQGTKVEIKR (SEQ. ID NO. 142) Ca Ka016-B5 MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVRPGESASISCKASQSLLHSGGGTYLNWFRQRPG  QSPQRLIYEVSKRDTGVPDRFSGSGSGTDFTLRITRVEADDTGIYYCGQNTQFPLTFGQGTKVEIK R (SEQ. ID NO. 143) Ca Ka016-B6MRFPSQLLGLLMLWIPGSSGGIVMTQTPLSLSV RPGETASISCRASQSLLYSDGNTYLFWFRQKPG QSPQRLMYRVSDRDTGVPDRFSGSGSGTDFTLT ISGVEADDAGIYYCGQATHYPLEFGQGTXVEIKR (SEQ. ID NO. 144) Ca Ka016-B7 MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVRPGESASISCKASQSLLHSGGGTYLNWFRQRPG  QSPQRLIYEVSKRDTGVPDRFIGSGAGTDFTLRISRVEADDAGVTYCGQGVQGPWTIGAGTKLELQ R (SEQ. ID NO. 145) Ca Ka016-B8MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSVSV SPGETASISCKASQSLLSHDGNTYLHWFRQKPG QSPQRLIYKVSNRDTGVPDRFSGSGSGTDFTLK ISRVEADDTGVYYCGQITQDPFTFGQGTKLEIKR (SEQ. ID NO. 146) Ca Ka016-B9 MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGETASISCRASQSLLHSNGNTYLFWFRQKPG  QSPQRLIKWVSNRDPGVPDRFGGSGSGTDFTLRISRVEADDAGIYYCGOGIOGPYTFSQGTKLEIK R (SEQ. ID NO. 147) Ca Ka016-B10MRFPSQFLGLLMLWIPGSSGDIAMTQTPLSLSV GPGETASITCKASQSLLHSNGKTYLFWFRQKPG QSPQRLIYLVSNRDPGVPDRFSGSGSGTDFTLT ISRVEADDAGIYYCGQATQTPPTFGAGTKLDLKR (SEQ. ID NO. 148) Ca Ka016-B11 MRFPSQLLGLLMLWIPGSSGDIVMAQTPLSLSVSPGEPASISCKASQSLLHSDGRTCLSWFRQKSG  QSPQRLIYEVSNRDTGVPDRFSGSGSGTDFTLRISRVEADDTGIYYCGQTVQFPLTFGQGTKLEIK R (SEQ. ID NO. 149) Ca Ka016-B12GQSPQRLIYKVSNRDPGVPDRFSGSGSGTDFTL RISRVEPEDVGVYYCGQGTLNPWTFGAGTKVELKR (SEQ. ID NO. 150) Ca Ka017-1 MRFPSQLLGLLMLWIPGSSGDVVMTQTPLSLSVSPGETASISCRASQSLLHSNGNTFLFWFRQ*PG  QSPQRLINFVSNRDPGVPDRFSGSGSGTDFTLR ISRVEADDAGIYYCGQGLLAPPTFGQGTKVEIR R (SEQ. ID NO. 151)NOTE: * INDICATES A STOP CODON Ca Ka017-2MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSV SPREPASISCKASQSLLRSNGNTYLYWFRQKPG QSPEGLIYRVSNRFTGVSDRFSGSGSGTDFTLR ISTVEADDAGVYYCGQATQFPSTFSQGTKLEIKR (SEQ. ID NO. 152) Ca Ka017-3 MRFPSQLLGLLMLWIPGSXGDIVLTQTPLSLSVSPGEPASISCKASQSLLMSNGITYLNWYRQRPG  QSPQXLIYKVSNRDTGVPDRFSGSGSGTDFTLRXSKVEADDTGIYYCGQDTQFPLTLGXGTHXEIK R (SEQ. ID NO. 153) Ca Ka017-5MRFPSQLLGLLMLWIPGSTGDIVMTQTPLSLSV SPGEPASIYCKASQSLLHSNGKTFLYWFRQKPG QSPQRLIYRVSNRDPGVPDRFSGSGSGTDFTLR ISRVEADDAGIYYCGQGIQDPTFGQGTKVEIKR(SEQ. ID NO. 154) Ca Ka017-6 MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSVSPREAASISCKASQSLLKSNGKTYFYWFRQKPG  QVSEGLIYKVSSRFTGVSDRFSGSGSGTDFTLRISRVEADDAGVYFCGQALQFPYTFSQGTKLDIK R (SEQ. ID NO. 155) Ca Ka017-10MRFPSQLLGLLMLWIPESGGDVVLTQTPPSLSL SPGETASISCKASRSLLKSDGSTYLDWYLQKPG QSPRLLIYLVSNRFSGVSDRFSGSGSGTDFTLT ISRVEADDAGVYYCGQGSRVPLTFGQGTKVEIKR (SEQ. ID NO. 156) Ca Ka017-11 MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSVSPGETASISCRASQSLLHRNGITYLSWFRQRPG  QSPQRLINLVSNRDPGVPDRFSGSGSGTDFTLRISRVEADDVGVYYCGHGLQTPYTFGQGTSLEIE R (SEQ. ID NO. 157) Ca Ka017-12MRFPSQLLGLLVLWIPGSSGDIVMTQTPLSLSV SPGETVSISCRASQSLLYSDGNFYLFWFRRKPGQSPQHLINLVSNRDPGVPDRFSGSGSGTDFTLR ISRVEADDAGVYYCGQGTQPPYTFSQGTKVEIKR (SEQ. ID NO. 158) Ca Ka017-13 MRFPSQLLGLLMLWIPESGGDVVLTQTPPSLSLSPGETASISCKASRSLLNSDGSTYLDWYLQKPG  QSPRLLIYLVSNRFSGVSDRFSGSGSGTDFTLTISRVEADDAGVYYCGQGSRVPLTFGQGTKVEIK R (SEQ. ID NO. 159) Ca Ka017-14MRFPSQLLGLLMLWIPGSSGDIVMAQTPLSLSV SPGETASISCRASQSLLHSNGITYLFWYRQKPG QSPQRLISMVFNRDPGVPDRFGGSGSGTDFTLR ISRVEADDAGLYFCGHGTQIPYSFSQGTKLEIKR (SEQ. ID NO. 160) Ca Ka017-16 MRFPSQLLGLLMLWIPGSSGDIVMTQTPLSLSISPGEPASISCKASQSLLHSGGDTYLNWFRQRPG  QSPQLLINRVSSRKKGVPDRFSGSGSGTEFTLRISRVEADDAGIYFCGQGTQFPYTFSQGTKLEIK R (SEQ. ID NO. 161) Ca Ka017-20MRFPSQLLGLLMLWIPGSGGDIVMTQTPPSLSV SPGEPASMSCKASQSLLHSNGNTYLYWFRQKPG QSPEALIYKVSNRFTGVSDRFSGSGSGTDFTLR INRVEADDVGVYYCGQGIQIPYTFSQGTKLEIKR (SEQ. ID NO. 162) Ca Ka017-23 MRFPSQLLGLLMLWIPGSTGEIVLTQTPLSLSVSPGESASTSCKASQSLLYSNGNTYLYWFRQKAG  QSPORVIYRVSKRDPGVPDRFSGSGSGTDFTLRISSVENDDAGVYYCGQGSEDPPTFGAGTKVELK R (SEQ. ID NO. 163) Ca Ka017-24MRFPSQLLGLLTLWIPGSTGDIVMTQTPLSLSV SPGEPASISCKASQSLLHSNGNTYLYWFRQKPGQSPQRLIYKVSNRDPGVPXRFSGSGSGTDFTLR VSXVEADDAGVYYCGQGVQDPFTFGQGTKLEIKR (SEQ. ID NO. 164)

Example 13 CDR-Grafting to Create Caninized Monoclonal Antibodies

To generate caninized antibody sequences from mouse anti-NGF antibodies,each murine variable heavy chain antibody gene sequence was separatelyaligned against 36 canine Ig germline variable heavy chain sequencesusing Vector NTI software. Eleven canine Ig germline variable heavychain sequences were derived from U.S. Pat. No. 7,261,890 B2, (Methodsfor Using Canine Immunoglobulin Variable Domains and CaninizedAntibodies), the contents of which are herein incorporated by reference,and 25 canine Ig germline variable heavy chain sequences were derivedfrom Table 14 (Canine Heavy Chain Variable Domain Sequences Derived fromCanine PBMC RNA). Each murine variable light chain gene sequence wasseparately aligned against 68 germline variable light chain sequences(derived from U.S. Pat. No. 7,261,890 B2) using Vector NTI software.Canine variable domain sequences having the highest overall homology tothe original murine sequences were selected for each heavy chain andlight chain sequence to provide the framework sequence. In silicoconstruction of complete CDR grafted antibodies was accomplished bysubstitution of canine variable domain CDR sequences with murine CDRsequences (derived from the subcloned anti-NGF antibody hybridoma mAbs).To identify residues in each sequence, the first amino acid in eachlisted sequence was defined as 1, and all remaining residues numberedconsecutively thereafter using Kabat numbering system.

The heavy chain CDR sequences from PR-1254972 were grafted in silicoonto canine 894 as follows: (1) One N-linked glycosylation pattern(N-{P}-S/T) was found in these proposed constructs. (2) Sixback-mutations (Q3H, V371, Q46E, D73N, T77N, R83K) were introduced tomake the 72.2 VH sequence. (3) One, two, three, four, five, or six ofthe back-mutations disclosed above could be introduced into 72.2 VH tomaintain antibody affinity to NGF after caninization of mAb 72.2. (4)One, two, three, four, five, or six of these back-mutations may besubstituted during subsequent affinity maturation of 72.2 VH. 72.3 VHwas generated by introducing the back-mutations in 72.2 VH with theaddition of H39Q back-mutation. 72.4 VH was generated by introducingback-mutations Q3H, H39Q, Q46E, D73N. The light chain CDR sequences fromPR-1254972 were grafted in silico onto canine 1001 as follows: (1) NoN-linked glycosylation pattern (N-{P}-S/T) was found in these proposedconstructs. (2) Four back-mutations (12V, V3L, Q45K, S59P) wereintroduced to make the 72.2 VL sequence. (3) One, two, three, or four ofthese back-mutations could be introduced into 72.2 VL to maintainantibody affinity to NGF after caninization of mAb 72.2. (4) One, two,three, or four of these back-mutations may be substituted duringsubsequent affinity maturation of 72.2 VL. 72.4 VL was generated byintroducing back-mutations Q45K, and S59P.

The heavy chain CDR sequences from PR-1254973 were grafted in silicoonto canine 894 as follows: (1) No N-linked glycosylation pattern(N-{P}-S/T) was found in these proposed constructs. (2) Eightback-mutations (T24A, M48I, V67A, L69V, T73K, N76S, V78A, A93T) wereintroduced to make the 73.2 VH sequence. (3) One, two, three, four,five, six, seven, or eight of these back-mutations could be introducedinto 73.2 VH to maintain antibody affinity to NGF after caninization ofmAb 73.2. (4) One, two, three, four, five, six, seven, or eight of theseeight back-mutations may be substituted during subsequent affinitymaturation of 73.2 VH. 73.4 VH was generated by introducingback-mutations T24A, T73K, A93T. The light chain CDR sequences fromPR-1254973 were grafted in silico onto canine 1034 as follows: (1) NoN-linked glycosylation pattern (N-{P}-S/T) was found in these proposedconstructs. (2) Eight back-mutations (I ID, V3Q, S22T, F36H, R46L, 148V,D60S, D70Q) were introduced to make the 73.2 VL sequence. (3) One, two,three, four, five, six, seven, or eight of these back-mutations could beintroduced into 73.2 VL to maintain antibody affinity to NGF aftercaninization of mAb 73.2. (4) One, two, three, four, five, six, seven,or eight of these eight back-mutations may be substituted duringsubsequent affinity maturation of 73.2 VL. 73.4 VL was generated byintroducing back-mutations I1D, V3Q, F36H, R46L, D60S, D70Q.

The heavy chain CDR sequences from PR-1254977 were grafted in silicoonto canine 894 as follows: (1) No N-linked glycosylation pattern(N-{P}-S/T) was found in these proposed constructs. (2) Eightback-mutations (T24A, Q38K, M481, R66K, V67A, T68S, L691, V78A) wereintroduced to make the 77.2 VH sequence. (3) One, two, three, four,five, six, seven, or eight of these back-mutations could be introducedinto 77.2 VH to maintain antibody affinity to NGF after caninization ofmAb 77.2. (4) One, two, three, four, five, six, seven, or eight of theseback-mutations may be substituted during subsequent affinity maturationof 77.2 VH. 77.3 VH was generated by introducing the back-mutations in77.2 VH with the addition of R94G back-mutation. 77.4 VH was generatedby introducing back-mutations T24A, Q38K, and R94G. The light chain CDRsequences from PR-1254977 were grafted in silico onto canine 997 asfollows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found inthese proposed constructs. (2) Four back-mutations (L2V, F36Y, R46L,S98G) were introduced to make the 77.2 VL sequence. (3) One, two, three,or four of these back-mutations could be introduced into 77.2 VL tomaintain antibody affinity to NGF after caninization of mAb 77.2. (4)One, two, three, or four of these back-mutations may be substitutedduring subsequent affinity maturation of 77.2 VL. 77.4 VL was generatedby introducing back-mutations F36Y and R46L.

The heavy chain CDR sequences from PR-1254981 were grafted in silicoonto canine 876 as follows: (1) No N-linked glycosylation pattern(N-{P}-S/T) was found in these proposed constructs. (2) Sixback-mutations (Q46E, G49A, T77N, R83K, L91Y, E93T) were introduced tomake the 81.2 VH sequence. (3) One, two, three, four, five, or six ofthese back-mutations could be introduced into 81.2 VH to maintainantibody affinity to NGF after caninization of mAb 81.2. (4) One, two,three, four, five, or six of these six back-mutations may be substitutedduring subsequent affinity maturation of 81.2 VH. 81.4 VH was generatedby introducing back-mutations Q46E, G49A, L91 Y, and E93T.

The light chain CDR sequences from PR-1254981 were grafted in silicoonto canine 1011 as follows: (1) No N-linked glycosylation pattern(N-{P}-S/T) was found in these proposed constructs (2) Fourback-mutations (V3L, A7T, F36Y, R46L) were introduced to make the 81.2VL sequence (3) One, two, three, or four of these back-mutations couldbe introduced into 81.2 VL to maintain antibody affinity to NGF aftercaninization of mAb 81.2. (4) One, two, three, or four of theseback-mutations may be substituted during subsequent affinity maturationof 81.2 VL. 81.4 VL was generated by introducing back-mutations A7T,F36Y, and R46L.

Alternatively, the heavy chain CDR sequences from PR-1254981 weregrafted in silico onto canine 1005 VH as follows: (1) No N-linkedglycosylation pattern (N-{P}-S/T) was found in these proposedconstructs. (2) Seven back-mutations (Q46E, T77N, F79Y, R83K, F91Y,V93T, K94R) were introduced to make the 81.5B VH sequence. (3) One, two,three, four, five, six, or seven of these back-mutations could beintroduced into 81.5B VH to maintain antibody affinity to NGF aftercaninization of mAb 81.5B. (4) One, two, three, four, five, six, orseven of these seven back-mutations may be substituted during subsequentaffinity maturation of 81.5B VH. 81.6B was generated by introducingback-mutations Q46E, F79Y, F91Y, and V93T. Variants 81.2B and 81.4B weregenerated by introducing A84K mutation to 81.5B and 81.6B, respectively.

The heavy chain CDR sequences from PR-1254982 were grafted in silicoonto canine 892 as follows: (1) No N-linked glycosylation pattern(N-{P}-S/T) was found in these proposed constructs. (2) Twelveback-mutations (13Q, 137V, M48L, 167L, T70S, A71K, G73N, N76S, H77Q,L78V, S79F, T93A) were introduced to make the 82.2 VH sequence. (3) One,two, three, four, five, six, seven, eight, nine, ten, eleven, or twelveof these back-mutations could be introduced into 82.2 VH to maintainantibody affinity to NGF after caninization of mAb 82.2. (4) One, two,three, four, five, six, seven, eight, nine, ten, eleven, or twelve ofthese back-mutations may be substituted during subsequent affinitymaturation of 82.2 VH. 82.4 VH was generated by introducingback-mutations 13Q, A71K, H77Q, S79F, and T93A. The light chain CDRsequences from PR-1254982 were grafted in silico onto canine 1034 asfollows: (1) No N-linked glycosylation pattern (N-{P}-S/T) was found inthese proposed constructs. (2) Ten back-mutations (I ID, V3Q, S22T,F36Y, Q45K, R46L, D60S, F71Y, T72S, Y87F) were introduced to make the82.2 VL sequence. (3) One, two, three, four, five, six, seven, eight,nine, or ten of these back-mutations could be introduced into 82.2 VL tomaintain antibody affinity to NGF after caninization of mAb 82.2. (4)One, two, three, four, five, six, seven, eight, nine, or ten of theseback-mutations may be substituted during subsequent affinity maturationof 82.2 VL. 82.3 VH was generated by introducing the back-mutations in82.2 VH with the addition of P44V back-mutation. 82.4 VL was generatedby introducing back-mutations 1 D, V3Q, F36Y, Q45K, R46L, D60S, F71Y,and Y87F.

Example 14 Isoelectric Point of Canine Framework Amino Acids

The heavy chain framework amino acids (i.e. non-CDR amino acids) of thecaninized IgG1 kappa antibodies yield a calculated isoelectric point ofless than 8.0. The light chain framework amino acids, when the lightchain is kappa, yield a calculated isoelectric point of less than 6.5.The isoelectric point of the caninized antibodies as a whole, i.e. heavyand light chain combined, due to the framework amino acids, and when thelight chain is kappa, is less than 8.0. In comparison, the frameworkamino acids of human IgG1 heavy chains typically yield isoelectricpoints of greater than 8.0. The framework amino acids of human kappalight chains typically yield isoelectric points of greater than 6.5. Theframework amino acids of whole human IgG1/k antibodies typically yieldisoelectric points of greater than 8.0.

Example 15 CDR-Grafting to Create Humanized Monoclonal Antibodies

Each murine variable heavy and variable light chain antibody genesequence (as set forth in Table 16) was separately aligned against 44human immunoglobulin germline variable heavy chain or 46 germlinevariable light chain sequences (derived from NCBI Ig Blast website whichis well known to those skilled in the art) using Vector NTI software.Human variable domain sequences having the highest overall homology tothe original murine sequences were selected for each heavy chain andlight chain antibody sequence to provide the framework (FW) 1, 2 and 3sequences for CDR-grafting purposes. Identification of a suitable humanvariable heavy and light chain FW4 region (also known as the “joining”region) was accomplished by separately aligning each murine heavy chainand light chain FW4 region with 6 human immunoglobulin germline joiningheavy chain and 5 germline joining light chain sequences in the NCBIdatabase. In silico construction of complete CDR grafted variabledomains was accomplished by substitution of human variable domain CDRsequences (derived from the NCBI website) with murine CDR sequences(derived from the murine antibodies) with addition of a FW4 region(derived from the NCBI website) to each 3′ end. Further humanization maybe accomplished by identification of back-mutations. Full length humanIgs may be produced by expressing the variable domains of eachCDR-grafted mAb with an in-frame human IgG constant domain. MouseAnti-NGF mAb CDRs grafted onto human Ig frameworks (CDR-grafted Anti-NGFAbs) produced are those listed in Table 17.

TABLE 17 Mouse Anti-NGF mAb CDRs Humanized byCDR Grafting onto Human Ig Frameworks NameSequence (CDRs are underlined) HU72 VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMF (CDR-GRAFTWVRQATGKGLEWVSTISDGGSYTYYTDNVKGRFTI VH3-13/JH5)SRENAKNSLYLQMNSLRAGDTAVYYCARDWSDSEG FAYWGQGTLVTVSS (SEQ ID NO: 165)Hu73 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMH (CDR-GRAFTWVRQAPGQGLEWMGRIDPYGGGTKHNEKFKRRVTM  VH1-18/JH6)TTDTSTSTAYMELRSLRSDDTAVYYCARSGYDYYF DVWGQGTTVTVSS (SEQ ID NO: 166)HU77 VH QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYIY (CDR-GRAFTWVROAPGQGLEWMGRIDPANGNTIYASKFQGRVTI VH1-69/JH6)TADKSTSTAYMELSSLRSEDTAVYYCARYGYYAYW GQGTTVTVSS (SEQ ID NO: 167) HU80 VHQVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIY (CDR-GRAFTWVRQAPGQGLEWMGRIDPANGNTIYASKFQGRVTM VH1-18/JH6)TTDTSTSTAYMELRSLRSDDTAVYYCARYGYYAYW GQGTTVTVSS (SEQ ID NO: 168) HU81 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFSNHYMY (CDR-GRAFTWVRQAPGKGLEWVGSISDGGAYTFYPDTVKGRFTI  VH3-15/JH1)SRDDSKNTLYLQMNSLKTEDTAVYYCTTEESANNG FAFWGQGTLVTVSS (SEQ ID NO: 169)HU82 VH QVTLKESGPVLVKPTETLTLTCTVSGFSLTGYNIN (CDR-GRAFTWIRQPPGKALEWLAMIWGYGDTDYNSALKSRLTIS  VH2-26/JH6)KDTSKSQVVLTMTNMDPVDTATYYCARDHYGGNDW YFDVWGQGTTVTVSS (SEQ ID NO: 170)HU72 VL DIVMTQTPLSLPVTPGEPASISCRSSQSIVQSNGN  (CDR-GRAFTTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGS  01/JK2)GSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGQ GTKLEIKR (SEQ ID NO: 171) HU73 VLDIQMIQSPSFLSASVGDRVSIICRASENIYSFLAW  (CDR-GRAFTLQKPGKSPKLFLYNANTLAEGVSSRFSGRGSGTDF  L22/JK2)TLTIISLKPEDFAAYYCQHHFGTPFTFGQGTKLEI KR (SEQ ID NO: 172) HU77VLDIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDGF  (CDR-GRAFT TYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGS  01/JK2)GSGTDFTLKISRVEAEDVGVYYCFESNYLFTFGQG TKLEIKR (SEQ ID NO: 173) HU80 VLDIVMTQTPLSLPVTPGEPASISCKSTKSLLNGDGF  (CDR-GRAFTTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGS  01/JK2)GSGTDFTLKISRVEAEDVGVYYCFESNYLFTFGQG TKLEIKR (SEQ ID NO: 174) HU81 VLDIVMTQTPLSLPVTPGEPASISCRSSQSILHSNGN  (CDR-GRAFTTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFSGS  01/JK2)GSGTDFTLKISRVEAEDVGVYYCFQGAHVPFTFGQ GTKLEIKR (SEQ ID NO: 175) HU82 VLDIQMTQSPSSLSASVGDRVTITCRASQDITNYLNW (CDR-GRAFTYQQKPGKAPKLLI YYTSRLHSGVPSRFSGSGSGT  08/JK2)DFTFTISSLQPEDIATYYCQQGKTLPRTFGQGTKL EIKR (SEQ ID NO: 176)

Example 16 Method for Constructing Full-Length Mouse/Canine Chimeric andCaninized Antibodies

Using conventional molecular biology techniques, a cDNA fragmentencoding the canine IgG1 constant region (which was obtained from theIMGT®, the International ImMunoGeneTics information system, which is theglobal reference in immunogenetics and immunoinformatics, created in byMarie-Paule Lefranc (Universite Montepellier 2 and CNRS)) wassynthesized and ligated to the 3′ end of each of the heavy chainvariable domains derived from murine anti-NGF monoclonal antibodiesPR-1254972, PR-1254973, PR-1254977, PR-1254981, PR-1254982. For thesesame anti-NGF mAbs, a cDNA fragment encoding the canine kappa constantregion obtained from U.S. Pat. No. 5,852,183 A, (Sequence ID No. 54) wassynthesized and ligated to the 3′ end of each of the light chainvariable domains. The complete canine IgG heavy chain constant domainnucleotide sequence and amino acid sequence is shown as SEQ ID NO: 51and SEQ ID NO: 52, respectively. The complete canine kappa light chainconstant domain nucleotide sequence and amino acid sequence is shown asSEQ ID NO: 53 and SEQ ID NO: 54, respectively. Complete heavy chain andlight chain chimeric cDNAs were ligated into the pHybE expressionplasmid; the sequences of these chimeric mAbs are in Table 18 below.

TABLE 18 Mouse/Canine Chimeric Antibody Sequences NameSequence (CDRs are underlined) PR-1290646 lightDVLMTQTPLSLPVSLGDQASISCRSSQSIVQ chain amino acidSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSG sequence VPDRFSGSGSGTDFTLKISREAEDLGVYYCFQGSHVPFTFGSGTKLEIKRNDAOPAVYLFQP SPDQLHTGSASVVCLLNSFYPKDIKVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSS TEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD (SEQ ID NO: 194) PR-1290646 heavy EVHLVESGGGLVKPGGFLILSCAASGFTFSDchain amino acid YYMFWIRQTPGKRLEWVATISDGGSYTYYTD sequenceNVKGRFTISRDNVKNNLYLQMSHLKSADTAM YYCARDWSDSEGFAYWGQGTLVTVSAASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEP VTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPV FNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFV DGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARG RAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDE DGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK (SEQ ID NO: 195) PR-1290654 lightDIQMTQSPASLSASVGETVTVTCRASENIYS chain amino acidFLAWHQQKQGKSPQLLVYNANTLAEGVPSRF sequence SGSGSGTQFSLKINSLQPEDFGSYYCQHHFGTPFTFGSGTKLEIKRNDAQPAVYLFQPSPDQ LHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYL SHELYSCEITHKSLPSTLIKSFQRSECQRVD(SEQ ID NO: 196) PR-1290654 heavy QVQLQQPGAELVKPGASVKLSCKASGYTFTNchain amino acid YWMHWVKQRPGQGLEWIGRIDPYGGGTKHNE sequenceKFKRKATVTADKSSSTAYILLSSLTSEDSAV YYCTRSGYDYYFDVWGTGTTVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPV TVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVF NECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVD GKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGR AHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDED GSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGV (SEQ ID NO: 197) PR-1290656 lightDVVLTQTPLSLPVNIGDQASISCKSTKSLLN chain amino acidGDGFTYLDWYLQKPGQSPQLLIYLVSNRFSG sequence VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFESNYLFTFGSGTKLEMKRNDAQPAVYLFQP SPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSS TEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD (SEQ ID NO: 198) PR-1290656 heavy EVQLQQSGAELVKPGASVKLSCTASGFNIKDchain amino acid TYIYWVKQRPEQGLEWIGRIDPANGNTIYAS sequenceKFQGKASITADTSSNTAYMQLSSLTSGDTAV YYCAGYGYYAYWGQGTTLTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVS WNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNEC RCTDTPPCPVPEPLGGPSVLIFPPKPKDILRTTRTPEVTCVVLDLGREDPEVQISWFVDGKE VHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHK PSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSY FLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK (SEQ ID NO: 199) PR-1290657 lightDVLMTQTPLSLPVSLGDQASISCRSSQSILH chain amino acidSNGNTYLEWYLQKPGQSPNLLIYRVSNRFSG sequence VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCRQGAHVPFTFGSGTKLEIKRNDAQPAVYLFQ PSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMS STEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD (SEQ ID NO: 200) PR-1290657 heavy EVQLVESGGGAVKPGGSLTLSCAASGFTFSNchain amino acid HYMYWVRQTPEKRLEEVASISDGGAYTFYPD sequenceTVKGRFTISRDNVNNNLYLQMRHLKSEDTAM YYCTREESANNGFAFWGQGTLVTVSAASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEP VTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPV FNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFV DGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARG RAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDE DGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGV (SEQ ID NO: 201)

The canine IgG1 constant region nucleotide sequence described above wasalso ligated to the 3′ end of each of the cDNAs encoding heavy chainvariable domains derived from caninized anti-NGF monoclonal antibodies72.2 VH, 72.3 VH, 72.4 VH, 73.2 VH, 73.4 VH, 77.2 VH, 77.3 VH, 77.4 VH,81.2 VH, 81.4 VH, 81.2B, 81.4B, 81.5B, 81.6B, 82.2 VH, 82.4 VH. Thecanine kappa light chain constant domain nucleotide sequence describedabove was also ligated to the 3′ end of each of the cDNAs encoding lightchain variable domains derived from caninized anti-NGF monoclonalantibodies 72.2 VL, 72.4, 73.2 VL, 73.4 VL, 77.2 VL, 77.4 VL, 81.2 VL,81.4 VL, 82.2 VL.

Full-length chimeric or caninized antibodies were transiently expressedin 293-6E cells by co-transfection of combinations of heavy and lightchain pHybE plasmids. Table 20 highlights all possible combination ofcaninized heavy and light chains that may be combined to produce acaninized antibody per the name in the table (Table 20). In Table 20,the heavy chain plasmids encoding caninized versions of murine heavychains are listed on the top line and proceed rightward. The light chainplasmids encoding caninized versions of murine light chains are listedon the left-hand column and proceed downward. At each point where theseboxes intersect, a name has been indicated to describe a potentialresulting caninized antibody.

Example 17 Caninized Monoclonal Antibody Expression and Purification

Selected heavy chain and light chain mouse/canine chimeric and caninizedantibody plasmids were co-transfected into 293-6e cells in suspensionand allowed to grow for 7-8 days. Cell supernatants were harvested,centrifuged, and filtered. For each expressed antibody, supernatant wasmixed with an equal volume of Pierce binding buffer to perform Protein ASepharose affinity chromatography according to manufacturer'sinstructions (GE Healthcare #17-1279-04). Although according to severalsources canine IgGs bind directly to Protein A moderately well (GEHealthcare Antibody Purification Handbook package insert; Scott, M. A.,et al., Vet Immunol-Immunopatho, 59:205, 1997; Warr, G. W and Hart, I.R., Am J Vet Res, 40:922, 1979; Thermo Scientific Pierce AntibodyProduction and Purification Technical Handbook) the monoclonal caninemAbs did not quantitatively bind to Protein A and therefore could not bepurified from supernatants without modification to the Protein Apurification methodology.

To allow quantitative binding of canine IgGs to Protein A, supernatantswere concentrated and mixed with an equal volume of Pierce bindingbuffer (Thermo #21007). To the concentrated and diluted supernatants,NaCl was added to a final concentration of 2.5 M. NaCl-adjustedsupernatant was loaded onto Protein A Sepharose by continuous over-nightloading, washed with Pierce binding buffer, and eluted using Pierceelution buffer (Thermo #21004). The eluates were neutralized by dropwiseaddition of 1M Tris pH 8.0; following this the neutralized antibodieswere dialyzed into PBS and amounts of antibody were quantifiedspectrophotometrically by OD₂8o- The amount purified was mathematicallydivided by the total volume of cell supernatant purified to determinethe overall estimated expression levels in μg/mL. The isolation andpurification of theses canine IgG1/k mAbs allowed analyticalcharacterization studies of the mAbs to be completed.

For purification of large-scale cell supernatants (10-15 L), cellsupernatants were concentrated, then mixed with Pierce binding buffer A(Thermo, catalog #21001) in a 1 to 1 ratio. To this mixture, 5 M NaClwas added to 1.3 M final concentration. The pH of the mixture wasadjusted to 8.5 with 10 N NaOH. The pH-adjusted cell supes were loadedonto a Protein A MabSelect SuRe (GE Healthcare, catalog #17-5438-03)chromatography column and eluted using two steps. The first step of theelution was performed using 20 mM Tris, 25 mM NaCl, pH 8.0, 7.4 ms/cm.Fractions containing antibodies were identified by OD₂80o and sizeexclusion chromatography. To quantitatively isolate the remainingantibody bound to the Protein A column, the second step elution wasperformed using Pierce elution buffer (Thermo, catalog #21004), pH 2.7,3.7 mS/cm, and fractions containing antibodies were identified by OD₂8oand size exclusion chromatography. All fractions containing antibodieswere neutralized using 2M Tris pH 8.5, and then dialyzed into PBS. Themethod employed to purify large volumes of cell supernatant containingcanine monoclonal antibodies (ex. 10-15 L) differs from the methodtypically employed to purify human antibodies from large volumes. Forhuman antibodies, Protein A purification is typically accomplished withcell supernatant binding conditions of pH 7.0 to 8.3 and 15 to 20 mS/cm,washing with similar conditions (1×PBS) and a 1 step elution of humanantibodies with 0.1 M acetic acid, 0.15 M sodium chloride, pH 2.7 at 15to 20 mS/cm or Thermo IgG elution buffer, pH 2.7, at 15 mS/cm.

Purified canine antibodies were analyzed by mass spectroscopy (MS) toconfirm the expressed antibody protein molecular weight matched theexpected weight based on amino acid sequence. In addition, canineantibodies were analyzed by size exclusion chromatography (SEC) todetermine the percent monomer. This data indicated that mouse/caninechimeric IgG1/k mAbs may be expressed transiently in 293-6e cells andare 81% or greater monomeric following purification. This data alsoindicated that caninized IgG1/k mAbs may be expressed transiently in293-6e cells and in most cases are 80% or greater monomeric followingpurification. In some cases, expression of protein may not be detectedand in some cases purified caninized mAb is between 24 and 34%monomeric. The data is summarized in Tables 19 and 20.

TABLE 19 Mouse/Canine Chimeric Monoclonal Antibody Characterization DataMoniker Estimated of Expression Mouse/ Level % Hybrid- Name of Canine InCell Mono- oma Mouse/Canine Chimeric Supernatants meric Moniker ChimericVersion Version (ug/mL) aAb PR- Mu72 Canine PR-1290646 3.2 97 1254972lgG1/k Chimera PR- Mu73 Canine PR-1290654 7 88.3 1254973 lgG1/k ChimeraPR- Mu77 Canine PR-1290656 0.3 82.4 1254977 lgG1/k Chimera PR- Mu81Canine PR-1290657 0.9 81 1254981 lgG1/k Chimera PR- Mu82 CaninePR-1290658 11.9 92.3 1254982 lgG1/k Chimera

TABLE 20 Production of Caninized Antibodies by Combinations of CaninizedHeavy and Light Chains Light chain Heavy chain 72.2 VH 72.3 VH 72.4 VH73.2 VH 73.4 VH 77.2 VH 72.2 VL 72VHv2/ 72.3 72VHv4/ 73.5 73VHv4/77VHv2/ 72VLv2 CaIgG1/k 72VLv2 CaIgG1/k 72VLv2 72VLv2 72.4 VL 72VHv2/72VHv3/ 72.4 73VHv2/ 73VHv4/ 77VHv2/ 72VLv4 72VLv4 CaIgG1/k 72VLv472VLv4 72VLv4 73.2 VL 72VHv2/ 72.5 72VHv4/ 73.2 73VHv4/ 77VHv2/ 73VLv2CaIgG/k 73VLv2 CaIgG1/k 73VLv2 73VLv2 73.4 VL 72VHv2/ 72VHv3/ 72VHv4/73VHv2/ 73.4 77VHv2/ 73VLv4 73VLv4 73VLv4 73VLv4 CaIgG/k 73VLv4 77.2 VL72VHv2/ 72.6 72VHv4/ 73.6 73VHv4/ 77VHv2/ 77VLv2 CaIgG/k 77VLv2 CaIgG1/k77VLv2 77VLv2 77.4 VL 72VHv2/ 72VHv3/ 72VHv4/ 73VHv2/ 73VHv4/ 77VHv2/77VLv4 77VLv4 77VLv4 77VLv4 77VLv4 77VLv4 81.2 VL 72VHv2/ 72.7 72VHv4/73.7 73VHv4/ 77VHv2/ 81VLv2 CaIgG/k 81VLv2 CaIgG1/k 81VLv2 81VLv2 81.4VL 72VHv2/ 72VHv3/ 72VHv4/ 73VHv2/ 73VHv4/ 77VHv2/ 81VLv4 81VLv4 81VLv481VLv4 81VLv4 81VLv4 82.2 VL 72VHv2/ 72VHv3/ 72VHv4/ 73VHv2/ 73VHv4/77VHv2/ 82VLv2 82VLv2 82VLv2 82VLv2 82VLv2 82VLv2 82.3 VL 72VHv2/ 72.872VHv4/ 73.8 73VHv4/ 77VHv2/ 82VLv3 CaIgG/k 82VLv3 CaIgG1/k 82VLv382VLv3 82.4 VL 72VHv2/ 72VHv3/ 72VHv4/ 73VHv2/ 73VHv4/ 77VHv2/ 82VLv482VLv4 82VLv4 82VLv4 82VLv4 82VLv4 Light chain Heavy chain 77.3 VH 77.4VH 81.2 VH 81.4 VH 82.2 VH 82.4 VH 72.2 VL 77.5 77VHv4/ 81.5 81VHv4/82.5 82VHv4/ CaIgG1/k 72VLv2 CaIgG1/k 72VLv2 CaIgG1/k 72VLv2 72.4 VL77VHv3/ 77VHv4/ 81VHv2/ 81VHv4/ 82VHv2/ 82VHv4/ 72VLv4 72VLv4 72VLv472VLv4 72VLv4 72VLv4 73.2 VL 77.6 77VHv4/ 81.6 81VHv4/ 82.6 82VHv4/CaIgG1/k 73VLv2 CaIgG1/k 73VLv2 CaIgG1/k 73VLv2 73.4 VL 77VHv3/ 77VHv4/81VHv2/ 81VHv4/ 82VHv2/ 82VHv4/ 73VLv4 73VLv4 73VLv4 73VLv4 73VLv473VLv4 77.2 VL 77.3 77VHv4/ 81.7 81VHv4/ 82.7 82VHv4/ CaIgG1/k 77VLv2CaIgG1/k 77VLv2 CaIgG1/k 77VLv2 77.4 VL 77VHv3/ 77.4 81VHv2/ 81VHv4/82VHv2/ 82VHv4/ 77VLv4 CaIgG1/k 77VLv4 77VLv4 77VLv4 77VLv4 81.2 VL 77.777VHv4/ 81.2 81VHv4/ 82.8CaIgG1/k 82VHv4/ CaIgG1/k 81VLv2 CaIgG1/k81VLv2 81VLv2 81.4 VL 77VHv3/ 77VHv4/ 81VHv2/ 81.4 82VHv2/ 82VHv4/81VLv4 81VLv4 81VLv4 CaIgG1/k 81VLv4 81VLv4 82.2 VL 77VHv3/ 77VHv4/81VHv2/ 81VHv4/ 82VHv2/ 82VHv4/ 82VLv2 82VLv2 82VLv2 82VLv2 82VLv282VLv2 82.3 VL 77.8 77VHv4/ 81.8 81VHv4/ 82.3 82VHv4/ CaIgG1/k 82VLv3CaIgG1/k 82VLv3 CaIgG1/k 82VLv3 82.4 VL 77VHv3/ 77VHv4/ 81VHv2/ 81VHv4/82VHv2/ 82.4 82VLv4 82VLv4 82VLv4 82VLv4 82VLv4 CaIgG1/k

TABLE 21 Caninized Monoclonal Antibody Characterization Data EstimatedExpression Level in % Cell Super- Mono- natants meric Name Moniker Lot(ug/mL) mAb 72.3 Canine PR-1313524 1804091 2.63 88.3 lgG1/k 72.4 CaninePR-1314949 1805928 1.6 81.5 lgG1/k 73.2 Canine PR-1313520 1810546 13.496.5 lgG1/k 73.4 Canine PR-1314950 1805932 1.8 90 lgG1/k 77.3 Canine N/AN/A 0.7 24.8 lgG1/k 77.4 Canine N/A N/A 1 34.6 lgG1/k 81.2 Canine N/A NomAb No mAb N/A lgG1/k detected detected 81.4 Canine N/A No mAb No mAbN/A lgG1/k detected detected 82.3 Canine PR-1313519 1810585 4.4 80.7lgG1/k 82.4 Canine PR-1313521 1816320 9.8 94.2 lgG1/k

Example 18 Affinity Analysis of Canine Antibodies

Purified mouse/canine chimeric antibodies and caninized antibodies wereanalyzed for affinity to canine NGF using a Biacore T100 instrument.Goat anti Canine IgG (Southern Biotech) was immobilized at 5000-10000 RUon a CM5 chip using an amine coupling procedure according to themanufacturer's instructions (Biacore). Canine NGF was injected at 50uL/min at a concentration range of 50-0.156 nM for the mouse/caninechimeric antibodies or 10-0.156 nM for the caninized antibodies. Theassociation rate was monitored for 5 min and the dissociation rate wasmonitored for 10-20 min. The chip surface was regenerated using 50-75 ul10 mM glycine pH 1.5 at a flow rate of 50-100 ul/min. Data was analyzedusing Biaevaluation T100 software version 2.0.2, software, GE HealthcareLife Sciences (Piscataway, N.J.). Overall affinity parametersestablished for mouse/canine chimeric antibodies is summarized in Table22 and for caninized antibodies in Table 23. This data indicates thatthe isolated mouse/canine chimeric anti-NGF mAbs have fast on-rates(from greater than 2×10⁶) and slow off-rates (from less than 3×10⁻³).The overall K_(D)s of the mouse/canine anti-NGF mAbs range from about1300 pM to 1.6 pM. This data also indicates that the isolated caninizedchimeric anti-NGF mAbs have fast on-rates (from greater than 6×10⁶) andslow off-rates (from less than 2×10⁻⁴). The overall K_(D)s of thecaninized anti-NGF mAbs range from about 42 pM to 1.2 pM.

TABLE 22 Affinity Parameters of Mouse/Canine Chimeric MonoclonalAntibodies to Canine NGF On-rate Off-rate Overall Name Moniker (l/M-S)(l/S) Affinity (M) Mu72 Canine PR- 2.9 × 10⁶ 3.8 × 10⁻³ 1.3 × 10⁻⁹ lg1/k Chimera 1290646 Mu73 Canine PR- 6.3 × 10⁶   9 × 10⁻³ 1.4 × 10⁻¹¹lg1/k Chimera 1290654 Mu77 Canine PR- 9.1 × 10⁶ 1.9 × 10⁻⁴ 2.1 × 10⁻¹¹lg1/k Chimera 1290656 Mu81 Canine PR- 4.2 × 10⁶ 3.5 × 10⁻⁴ 8.2 × 10⁻¹¹lg1/k Chimera 1290657 Mu82 Canine PR- 8.7 × 10⁶ 1.4 × 10⁻⁵ 1.6 × 10⁻¹²lg1/k Chimera 1290658

TABLE 23 Affinity Parameters of Caninized Monoclonal Antibodies toCanine NGF On-rate Off-rate Overall affinity Name (l/M-s) (l/s) (M) 73.2canine lgG1/k Expt 1 6.3 × 10⁶ 2.8 × 10⁻⁴ 4.4 × 10⁻¹¹ PR-13113520 Expt 26.9 × 10⁶ 2.9 × 10⁻⁴ 4.2 × 10⁻¹¹ Average 6.6 × 10⁶ 2.9 × 10⁻⁴ 4.3 ×10⁻¹¹ 82.3 canine lgG1/k Expt 1 8.2 × 10⁶   2 × 10⁻⁵ 2.4 × 10⁻¹²PR-13113519 Expt 2 8.5 × 10⁶ 1.3 × 10⁻⁵ 1.6 × 10⁻¹² Average 8.4 × 10⁶1.7 × 10⁻⁵   2 × 10⁻¹² 82.4 canine lgG1/k Expt 1 8.6 × 10⁶ 1.1 × 10⁻⁵1.2 × 10⁻¹² PR-13113521 Expt 2 7.7 × 10⁶ 1.2 × 10⁻⁵ 1.5 × 10⁻¹² Average8.2 × 10⁶ 1.2 × 10⁻⁵ 1.4 × 10⁻¹²

Example 19 Characterization of Canine Antibodies by the TF-1 CellProliferation Potency Assay

Purified mouse/canine chimeric antibodies and caninized antibodies werecharacterized using the TF-1 Cell Proliferation Potency Assay (describedpreviously) using 70 pM canine NGF in the assay. The summarized potencydata is in Tables 20 and 21. The data shows that in the presence of 70pM canine NGF, all of the mouse/canine chimeric anti-NGF antibodiesdisplay sub-nM potencies, and all display potencies of less than 50 pM.The data shows that in the presence of 70 pM canine NGF, some of thecaninized anti-NGF antibodies have no neutralization potency on 70 pMcanine NGF. Some caninized mAbs have sub-nM potencies, and some havepotencies of less than 20 pM.

TABLE 24 Potency of Mouse/Canine Chimeric NGF Monoclonal Antibodies onCanine NGF-induced TF-1 Ce Proliferation Name Moniker Lot IC₅₀ (nM) Mu72Canine PR-1290646 1785614 0.041 lgG1/k Chimera Mu73 Canine PR-12906541785658 0.008 lgG1/k Chimera Mu77 Canine PR-1290656 1785699 0.028 lgG1/kChimera Mu81 Canine PR-1290657 1778832 0.012 lgG1/k Chimera Mu82 CaninePR-1290658 1785732 0.007 lgG1/k Chimera

TABLE 25 Potency of Caninized NGF Monoclonal Antibodies on CanineNGF-Induced TF-1 Cell Proliferation (N/A = not applicable) Name MonikerLot IC₅₀ (nM) 72.3 Canine lgG1/k PR-1313524 1804091 0 72.4 Canine lgG1/kPR-1314949 1805928 0 73.2 Canine lgG1/k PR-1313520 1810546 0.422 73.4Canine lgG1/k PR-1314950 1805932 0 77.3 Canine lgG1/k N/A N/A 0.625 77.4Canine lgG1/k N/A N/A 0 82.3 Canine lgG1/k PR-1313519 1810585 0.017 82.4Canine lgG1/k PR-1313521 1816320 0.016

Example 20 Characterization of Solubility and Stability of CaninizedAnti-NGF Antibodies

Stock solutions of two caninized anti-NGF antibodies (73.2 canine IgG1/kand 82.4 canine IgG1/k) were obtained. The antibodies were formulated inphosphate buffer saline (PBS) at concentrations below 5 mg/ml (PBScontains, but is not limited to, the following ingredients: 15 mMphosphate buffer and 150 mM sodium chloride at pH 7.4).

Solubility:

The solubility of the caninized antibodies at high concentrations in PBSwere evaluated by concentrating the antibodies with Amicon 30K molecularweight cutoff centrifuge spin filters.

The final concentrations were determined by UV absorbance.

At room temperature, 73.2 canine IgG1/k was soluble to at least 54 mg/mland 82.4 canine IgG1/k was soluble to at least 83 mg/ml. When stored at5° C. for 5 hours at those concentrations, 73.2 canines IgG1/k formed agel layer at the bottom of the container while 82.4 canines IgG1/kremained as a uniform solution. When re-equilibrated to roomtemperature, 73.2 canines IgG1/k became a uniform solution. When 73.2canines IgG1/k were diluted to 27 mg/ml, it remained as a uniformsolution at 5° C.

In comparison, adalimumab, a human antibody, demonstrated a solubilityof at least 150 mg/ml at 5° C. and at room temperature. This wasobserved in a formulation with a pH of 7 and with a sodium chlorideconcentration of 150 mM. The observations are described in Table 26

TABLE 26 Solubility of 73.2 canine lgG1/k, 82.4 canine lgG1/k and humanantibody adalimumab in PBS. Room temperature Observations Antibodysolubility (mg/ml) when placed at 5° C. 73.2 canine ≧54 Gel layer formedat lg1/k container bottom* 82.4 canine ≧83 Remained as solution lgG1/kadalimumab ≧150 Remained as solution *returned to uniform solution whenbrought back to room temperature; when diluted to 27 mg/ml, remained asuniform solution at 5° C.

The solubility of 73.2 canine IgG1/k 82.4 canine IgG1/k was alsoevaluated in 15 mM histidine buffer pH 6.0. This is a buffer typicallyused to formulate human therapeutic antibodies. The PBS buffercomprising the stock solutions of 73.2 canine IgG1/k and 82.4 canineIgG1/k were exchanged with the histidine buffer using Amicon 3 OKmolecular weight cutoff centrifuge spin filters. Following bufferexchange, the antibodies exhibited white precipitation and solubilitiesof less than 2 mg/ml at room temperature, as determined by UVabsorbance. In comparison, the human antibody adalimumab was observed toreach a concentration of at least 150 mg/ml in 15 mM histidine buffer pH6.0 at room temperature. These observations are summarized in Table 27.

TABLE 27 Solubility of anti-NGF caninized antibodies 73.2 canine lgG1/k,82.4 canine lgG1/k and human antibody adalimumab in 15 mM histidinebuffer pH 6.0. Room temperature Antibody solubility (mg/ml) Observations73.2 canine lgG1/k <2 White precipatate observed 82.4 canine lgG1/k <2White precipatate observed adalimumab ≧150 Remained as solution

Freeze-Thaw Stability

An assessment of the freeze-thaw (FT) stability of 73.2 canine IgG1/kand 82.4 canine IgG1/kin PBS, and after dilution with PBS to 1 mg/ml,was performed. Both antibodies were frozen at −80° C. for at least 4hours. They were then thawed in a 30° C. water bath (this constitutesone freeze-thaw cycle). Stability was assessed for four freeze-thawcycles by size exclusion HPLC (SEC). The freeze-thaw analysis issummarized in Table 28.

TABLE 28 Freeze-thaw stability of 73.2 canine lgG1/k and 82.4 caninelgG1/k at 1 mg/ml in PBS. Percentage Species Post Post Post AntibodySpecies Pre-FT FT#1 FT#2 FT#4 73.2 canine Monomer 97.4 97.3 97.3 97.2lgG1/k Aggregate 1.7 1.8 1.8 1.8 Fragment 0.9 0.9 0.9 1 82.4 canineMonomer 96.6 96.6 96.6 96.1 lgG1/k Aggregate 2.9 2.9 2.9 3.2 Fragment0.5 0.5 0.5 0.7

Storage Stability and Accelerated Stability:

The stability of 73.2 canine IgG1/k and 82.4 canine IgG1/k whenformulated at a concentration of 10 mg/mL and within a pH range of 5 to8 and at low (˜7.5 mM) and high (˜>150 mM) ionic strengths was assessed.Stability at these conditions was assessed by monitoring the stabilityof the antibodies in the following buffers and salt concentrations: (A)15 mM acetate pH 5; (B) 15 mM acetate pH 5+150 mM NaCl; (C) 15 mMhistidine pH 6+150 mM NaCl; (D) 15 mM phosphate pH 7.4; (E) PBS pH 7.4;(F) 15 mM Tris pH 7.5; (G) 15 mM Tris pH 8.0. Sodium azide (0.02%) wasadded to all buffers as an anti-microbial agent.

Stock solutions of 73.2 canine IgG1/k and 82.3 canine IgG1/kin PBS wereconcentrated up to 15 mg/ml using 3 OK molecular weight cutoffcentrifuge spin filters. They were then dialyzed against the bufferslisted above for 18 hours using mini-dialysis 1 kD molecular weightcut-off dialysis tubes (GE Healthcare). Following dialysis, samples werediluted with the respective buffers to a final concentration of 10mg/ml. 150 μl of each sample was aliquoted to cryovials which were thenstored at 40° C. or 5° C. Samples were analysed at time=0 hours (TO), at7 days (T7d), and at 21 days (T21d) and stability was assessed by SEC.

After 21 days at 40° C., accelerated stability testing showed that 73.2canine IgG1/k and 82.3 canines IgG1/k have much greater fragmentation atpH values below 7.4 than at pH values above 7.4. In comparison, thehuman antibody adalimumab, exhibited less fragmentation within the pHrange 4 to 8 over 21 days at 40° C. In particular, the fragmentation ofadalimumab at pH 6 was much less than the fragmentation of 73.2 canineIgG1/k or 82.4 canine IgG1/k at pH 6. Also, adalimumab at the higherstress condition of pH 4 showed equal or less fragmentation compared to73.2 canine IgG1/k or 82.4 canine IgG1/k at the lower stress conditionof pH 5. The results of the stability analyses and fragmentationprofiles are shown, respectively, in Tables 29 and 30. These datasuggest that canine IgG1/k monoclonal antibodies have a differentdegradation profile compared to that of human IgG1/k monoclonalantibodies. Specifically, the fragmentation appears to be more extensivefor canine IgG1/k antibodies than for human antibodies at pH 6 andbelow.

TABLE 29 Stability data from SEC for 73.2 canine IgG1/k, 82.4 canineIgG1/k and human antibody adalimumab in different formulations at 7 and21 days at 5° C. and at 40° C. Percentage Percentage Percentage MonomerAggregate Fragment Buffer T0 T7d T21d T0 T7d T21d T0 T7d T21d 73.2canine IgG1/k at 5° C. A (pH 5) 94.6 91.4 90.3 2.9 3.1 3.4 2.6 5.5 6.2 B(pH 5) 95.2 98.2 98.2 3.4 0.4 0.5 1.4 1.4 1.3 C (pH 6) 93.9 98.0 97.84.4 0.5 0.7 1.8 1.5 1.5 D (pH 7.4) 94.3 97.9 97.7 4.7 0.6 0.8 1.0 1.51.5 E (pH 7.4) 94.5 97.9 97.8 4.5 0.5 0.8 1.0 1.6 1.5 F (pH 7.5) 94.598.0 97.8 4.0 0.5 0.8 1.6 1.5 1.5 G (pH 8.0) 93.7 97.8 97.6 4.6 0.7 0.91.7 1.5 1.5 73.2 canine IgG1/k at at 40° C. A (pH 5) 94.6 81.3 79.0 2.94.5 5.1 2.6 14.2 15.9 B (pH 5) 95.2 92.1 90.9 3.4 0.6 1.2 1.4 7.4 7.8 C(pH 6) 93.9 94.4 91.6 4.4 0.6 1.1 1.8 5.0 7.3 D (pH 7.4) 94.3 97.5 96.64.7 0.9 1.6 1.0 1.5 1.8 E (pH 7.4) 94.5 98.0 97.3 4.5 0.6 1.1 1.0 1.41.6 F (pH 7.5) 94.5 97.5 96.3 4.0 0.9 1.8 1.6 1.6 1.9 G (pH 8.0) 93.797.0 95.2 4.6 1.2 2.6 1.7 1.8 2.2 82.4 canine IgG1/k at 5° C. A (pH 5)96.7 98.1 98.7 2.3 1.3 .8 1.0 0.5 0.5 B (pH 5) 96.3 97.2 97.3 2.4 2.32.3 1.3 0.5 0.4 C (pH 6) 97.0 97.1 97.1 2.6 2.3 2.3 0.4 0.6 0.6 D (pH7.4) 96.4 97.0 97.1 2.5 2.4 2.5 1.0 0.5 0.4 E (pH 7.4) 96.7 96.8 96.82.8 2.5 2.6 0.5 0.7 0.6 F (pH 7.5) 96.8 97.1 96.9 2.9 2.5 2.5 0.2 0.50.6 G (pH 8.0) 96.6 96.9 96.9 2.5 2.5 2.5 0.9 0.6 0.6 82.4 canine IgG1/kat 40° C. A (pH 5) 96.7 93.1 87.8 2.3 2.8 4.2 1.0 4.1 8.0 B (pH 5) 96.393.3 91.3 2.4 2.5 3.1 1.3 4.3 5.6 C (pH 6) 97.0 94.1 92.5 2.6 2.3 2.70.4 3.5 4.8 D (pH 7.4) 96.4 93.5 93.8 2.5 3.4 3.3 1.0 3.1 2.9 E (pH 7.4)96.7 95.1 93.6 2.8 2.4 2.6 0.5 2.5 3.8 F (pH 7.5) 96.8 93.4 923.6 2.92.6 3.0 0.2 4.0 0.5 G (pH 8.0) 96.6 94.8 92.7 2.5 2.8 3.5 0.9 2.4 0.4adalimumab at 40° C. pH 4 99 98 95 <1 <2 0.5 <1 <2 4.5 6 99 99 99 <1 <10.3 <1 <1 0.7 8 99 98 98 <1 <2 1.2 <1 <2 0.8

TABLE 30 Fragmentation profile from SEC for 73.2 canine lgG1/k, 82.4canine lgG1/k and human antibody adalimumab in different formulations at21 days and at 40° C. Increase in Percent Fragmentation over 21 days atAntibody Buffer 40° C. 73.2 canine lgG1/k A (pH 5) 13.3 B (pH 5) 6.4 C(pH 6) 5.5 D (pH 7.4) 0.8 E (pH 7.4) 0.6 F (pH 7.5) 0.3 G (pH 8.0) 0.582.4 canine lgG1/k A (pH 5) 7.0 B (pH 5) 4.3 C (pH 6) 4.4 D (pH 7.4) 1.9E (pH 7.4) 3.3 F (pH 7.5) 0.3 G (pH 8.0) −0.5 adalimumab pH 4 <4.5 pH 6<0.7 pH 8 <0.8

Example 21 Canine Single Dose PK Study and Antigen Bridging Assay for PKSerum Sample Analysis

The serum levels of 73.2 canine IgG1/k and 82.4 canine IgG1/k wereanalyzed following a single dose of 4.5 mg/kg (intravenous orsubcutaneous) in mongrel dogs. Following the injection, 13 samples ofvenous blood were collected over 672 hours. Blood samples were allowedto dot and the serum removed for antibody quantitation.

An NGF bridging assay was developed to quantitate canine anti-NGF mAbsin serum. Streptavidin-coated 96-well plates (MSD #L1 ISA-1) wereblocked with Blocker A (MSD #R93BA-4). Canine anti-NGF antibody presentin serum (or in PBS) was mixed with equimolar ratios of biotin-taggedNGF and Sulfo-tagged NGF (Sulfo Reagent MSD #R91AN-1) and incubated toform an NGF+antibody complex. The final concentration of thebiotin-tagged NGF and sulfo-tagged NGF in the assay was between 1-2 nM.NGF-antibody complexes were added to the streptavidin-coated plate andallowed to bind for 60 minutes. Following incubation, plates were washedwith PBS plus 0.05% Tween-20, and bound NGF-antibody complexes weredetected in Read Buffer T (MSD #R92TC-1) on an MSD SECTOR Imager 6000.Data was quantitated to estimate the total amount of antibody in μg/mLof a sample liquid and is provided below in Tables 27-30.

TABLE 31 Serum Concentrations of 82.4 canine lgG1/k Following a SingleSubcutaneous Dose Dog # Hours 1073305 1072602 1072104 1072306 1072105post injection ug/mL   0 0.0 0.0 0 0 0.1   0.25 2.6 0.4 0.0 2.24 0.1   19.0 2.2 0.9 9.07 0.4   8 31.5 17.7 13.8 32.55 12.8  12 41.1 20.5 18.733.31 24.5  24 42.2 25.6 23.2 35.73 23.8  48 52.3 37.2 36.3 41.35 35.3 72 51.2 41.1 34.4 38.91 36.8 144 47.1 42.6 35.2 33.46 36.7 240 39.032.4 29.0 26.03 31.7 336 30.9 28.2 24.1 19.99 26.8

TABLE 32 Serum Concentrations of 82.4 canine lgG1/k Following a SingleIntravenous Dose Dog # Hours post 1072705 1073804 1073303 10739031074502 injection ug/mL  0 0 0 0 0 0  0.25 102.6 83.1 84.0 80.1 81.7  1106.7 70.1 87.4 74.1 81.9  8 88.4 65.7 68.1 67.4 68.9  12 91.5 61.6 62.662.1 59.3  24 87.4 60.0 57.2 53.9 53.0  48 76.6 49.9 52.6 50.9 50.2  7260.3 49.1 46.0 40.8 44.6 144 45.7 43.1 36.1 35.7 36.0 240 37.2 34.4 32.624.3 32.6 336 31.7 32.7 24.8 20.6 20.1 504 23.5 17.4 18.5 12.2 12.8 67215.2 10.7 12.5 7.3 8.3

TABLE 33 Serum Concentrations of 73.2 canine lgG1/k Following a SingleSubcutaneous Dose Hours Dog # post 1072607 1074307 1072606 1074503injection ug/mL  0 0 0 0 0  0.25 0 0 2.1 0  1 1.0 5.9 4.7 0.0  8 18.631.3 18.7 7.4  12 22.7 32.5 21.3 8.7  24 26.8 33.2 24.2 12.0  48 33.735.9 28.4 16.2  72 35.0 37.6 30.7 19.4 144 34.9 37.4 30.2 21.8 240 31.631.8 26.8 22.0 336 24.4 24.7 22.6 16.5 504 15.3 14.3 13.8 10.6 672 6.89.2 5.1 5.4

TABLE 34 Serum Concentrations of 73.2 canine lgG1/k Following a SingleIntravenous Dose Hours Dog # post 1072804 1073304 1072604 injectionug/mL  0 0 0 0  0.25 93.6 33.2 108.5  1 86.3 30.8 103.1  8 76.9 22.185.4  12 72.1 21.4 80.5  24 63.0 17.1 68.0  48 54.6 14.6 56.8  72 49.413.5 50.8 144 41.4 10.2 41.4 240 35.4 8.9 30.8 336 30.5 6.3 20.9 50422.2 4.0 3.4 672 14.8 3.4 0.0

Example 22 Pharmacokinetic Analysis of Serum Concentration Data

Pharmacokinetic parameters for both intravenous (IV) and subcutaneous(SC) dosing routes were calculated for each animal using WinNonlinsoftware (Pharsight Corporation, Mountain View, Calif.) bynoncompartmental analysis. Other calculations, e.g. mean, standarddeviation (SD), and percent subcutaneous bioavailability (F: %) werecarried out using Microsoft Excel software (Microsoft CorporationRedmond, Wash.). The data is shown in Table 35 and 36.

TABLE 35 Pharmacokinetic Analysis of 73.2 canine IgG1/k Following aSingle Intravenous Dose IV SC T½ Vss CI T½ Cmax Tmax (day) (mL/kg)(mL/h/kg) (day) (ug/mL) (day) % F 14.8* 71 0.15 8.0* 31.3 4.8 51*Harmonic Mean

TABLE 36 Pharmacokinetic Analysis of 82.4 canine IgG1/k Following aSingle Intravenous Dose IV SC T½ Vss CI T½ Cmax Tmax (day) (mL/kg)(mL/h/kg) (day) (ug/mL) (day) % F 10.9* 73 0.19 11.6* 41.9 3.0 94*Harmonic Mean

The data indicates that canine mAbs 73.2 and 82.4 have a half-life ofabout 8 to about 15 days when dosed IV or SC, suggesting that thesemolecules exhibit mammalian antibody-like half-lives and overall PKparameters.

Example 23 ELISA for Titering Canine Antibodies

To quantitate canine antibodies in cell supernatants (or other liquids),high-binding EIA plates (Costar #9018) were coated with polyclonal goatanti-dog IgG antibodies (Rockland #604-1102) at 4 μg/ml in PBS. Afterblocking with 2% non-fat milk in PBS, canine monoclonal antibodies wereadded to the plates and the plates were washed with PBS plus 0.05%Tween-20. Bound canine mAbs were detected with HRP-tagged goat anti-dogIgG antibodies (Rockland #604-1302) at 0.1 μg/ml. Plates were washedwith PBS plus 0.05% Tween-20. Canine mAbs were detected by addition ofTMB substrate (Neogen #308177), and the reaction was stopped with INHC1. Bound canine antibodies were quantitated by absorption at 450 nM toestimate the total amount of antibody in μg/mL of a sample liquid.

The present disclosure incorporates by reference in their entiretytechniques well known in the field of molecular biology. Thesetechniques include, but are not limited to, techniques described in thefollowing publications:

-   Ausubel, F. M. et al. eds., Short Protocols in Molecular Biology    (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).-   Lu and Weiner eds. Cloning and Expression Vectors for Gene Function    Analysis (2001)-   BioTechniques Press. Westborough, Mass. 298 pp. (ISBN    1-881299-21-X).-   Kontermann and Dubel eds., Antibody Engineering (2001)    Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).-   Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An    Introduction To Genetic Engineering (3d Ed. 1985) Blackwell    Scientific Publications, Boston. Studies in Microbiology; V.2:409    pp. (ISBN 0-632-01318-4).-   Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d    Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN    0-87969-309-6).-   Winnacker, E. L. From Genes To Clones: Introduction To Gene    Technology (1987) VCH Publishers, NY (translated by Horst    Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).

All references, publications, and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

Although a number of embodiments, aspects and features have beendescribed above, it will be understood by those skilled in the art thatmodifications and variations of the described embodiments and featuresmay be made without departing from the present disclosure or thedisclosure as defined in the appended claims.

1-20. (canceled)
 21. An isolated antigen binding protein comprising: (a) heavy chain variable region (VH) comprising: (i) Complementarity Determining Region 1 (CDR1) selected from the group consisting of: SEQ ID NO. 55; SEQ ID NO. 61; SEQ ID NO. 67; and SEQ ID NO. 73; (ii) Complementarity Determining Region 2 (CDR2) selected from the group consisting of: SEQ ID NO. 56; SEQ ID NO. 62; SEQ ID NO. 68; and SEQ ID NO. 74; and; (iii) Complementarity Determining Region 3 (CDR3) selected from the group consisting of: SEQ ID NO. 57; SEQ ID NO. 63; SEQ ID NO. 69; SEQ ID NO. 75; and variants thereof having one or more conservative amino acid substitutions; and (b) a light chain variable region (VL) comprising: (i) CDR1 selected from the group consisting of: SEQ ID NO. 58; SEQ ID NO. 64; SEQ ID NO. 70; and SEQ ID NO. 76; (ii) CDR2 selected from the group consisting of: SEQ ID NO. 59; SEQ ID NO. 65; SEQ ID NO. 71; and SEQ ID NO. 77; and (iii) CDR3 selected from the group consisting of: SEQ ID NO. 60; SEQ ID NO. 66; SEQ ID NO. 72; and SEQ ID NO. 78; and variants thereof having one or more conservative amino acid substitutions, wherein the isolated antigen binding protein specifically binds to Nerve Growth Factor (NGF).
 22. The isolated antigen binding protein of claim 21 wherein (a) the VH region comprises: (i) CDR1 comprising SEQ ID NO. 55 (ii) CDR2 comprising SEQ ID NO. 56; and (iii) CDR3 comprising SEQ ID NO. 57; and (b) the VL region comprises: (i) CDR1 comprising SEQ ID NO. 58 (ii) CDR2 comprising SEQ ID NO. 59; and (iii) CDR3 comprising SEQ ID NO.
 60. 23. The isolated antigen binding protein of claim 21 wherein (a) the VH region comprises: (i) CDR1 comprising SEQ ID NO. 61 (ii) CDR2 comprising SEQ ID NO. 62; and (iii) CDR3 comprising SEQ ID NO. 63; and (b) the VL region comprises: (i) CDR1 comprising SEQ ID NO. 64 (ii) CDR2 comprising SEQ ID NO. 65; and (iii) CDR3 comprising SEQ ID NO.
 66. 24. The isolated antigen binding protein of claim 21 wherein (a) the VH region comprises: (i) CDR1 comprising SEQ ID NO. 67 (ii) CDR2 comprising SEQ ID NO. 68; and (iii) CDR3 comprising SEQ ID NO. 69; and (b) the VL region comprises: (i) CDR1 comprising SEQ ID NO. 70 (ii) CDR2 comprising SEQ ID NO. 71; and (iii) CDR3 comprising SEQ ID NO.
 72. 25. The isolated antigen binding protein of claim 21 wherein (a) the VH region comprises: (i) CDR1 comprising SEQ ID NO. 73 (i) CDR2 comprising SEQ ID NO. 74; and (i) CDR3 comprising SEQ ID NO. 75; and (b) the VL region comprises: (i) CDR1 comprising SEQ ID NO. 76 (i) CDR2 comprising SEQ ID NO. 77; and (i) CDR3 comprising SEQ ID NO.
 78. 26. The antigen binding protein of claim 21, wherein said antigen binding protein reduces, inhibits, or neutralizes the biological function of NGF.
 27. The antigen binding protein of claim 21 wherein said antigen binding protein is selected from the group consisting of: an immunoglobulin molecule, disulfide linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, humanized mAb, caninized mAb, canine mAb, feline mAb, felinized mAb, equine mAb, equinized mAb, multispecific antibody, a Fab, a dual specific antibody, a DVD-Ig, a Fab′, a bispecific antibody, a F(ab′)2, and a Fv.
 28. The antigen binding protein of claim 27, wherein said antigen binding protein is a monoclonal antibody.
 29. The antigen binding protein of claim 27, wherein the antigen binding protein is a chimeric antibody.
 30. The antigen binding protein of claim 27, wherein said antigen binding protein is a caninized mAb.
 31. The antigen binding protein of claim 27 wherein said antigen binding protein is a humanized mAb.
 32. A pharmaceutical composition comprising a therapeutically effective amount of the antigen binding protein of claim 21 and a pharmaceutically acceptable carrier, diluent or excipient.
 33. A method of inhibiting NGF comprising administration of the composition of claim
 32. 34. A method of reducing NGF activity in a subject suffering from an NGF-related disorder comprising administration of the composition of claim 32 to a subject.
 35. The method of claim 34 wherein the subject comprises a human, a canine or a feline.
 36. The method of claim 35 wherein the subject is canine.
 37. A method of detecting NGF in a sample comprising: (a) incubating a sample comprising NGF in the presence of an antigen binding protein according to claim 1; and (b) detecting the antigen binding protein which is bound to NGF in the sample. 